Tripeptide derivatives and their application in assaying enzymes

ABSTRACT

Tripeptide derivatives having the formula ##STR1## wherein R 1  is alkanoyl or ω-aminoalkyanoyl, phenylalkanoyl or p-aminophenylalkanoyl, cyclohexylcarbonyl or 4-aminomethyl-cylcohexylcarbonyl, benzoyl optionally substituted with methyl, amino, or halogen in the o- or p-position, alkoxy carbonyl, benzyloxy carbonyl optionally substituted with methoxy, methyl, or chlorine in the p-position, alkylsulfonyl, phenylsulfonyl or naphthylsulfonyl, R 2  is straight-chained or branched alkyl, hydroxyalkyl, alkoxyalkyl, benzoxyalkyl, ω-carboxy-alkyl, ω-alkoxy carbonylalkyl, ω-benzyloxycarbonylalkyl, cyclohexyl, cyclohexylmethyl, 4-hydroxycyclohexylmethyl, phenyl, benzyl, 4-hydroxybenzyl or imidazol-4-yl-methyl, R 3  is hydrogen or alkyl, R 4  is hydrogen, methyl or ethyl, and R 5  is an amino group substituted with aromatic or heterocyclic radicals, R 5  being capable of being split off by enzymatic hydrolysis to form a colored or fluorescent product H-R 5 . The tripeptide derivatives of formula I are used for assaying certain enzymes, and in particular, factor Xa. Enzyme-bearing materials are reacted with the said tripeptide derivatives. The quantity of split product H-R 5  released by the enzymatic action on the tripeptide derivative is determined photometrically, spectrophotometrically, fluorescence-spectrophotometrically, or electrochemically. The quantity of released split product H-R 5  per time unit is proportional to the quantity of enzyme present in the starting material.

TECHNICAL FIELD

The present invention relates to tripeptide derivatives which are veryeasily split by certain enzymes of the enzyme class 3.4.21., moreparticularly factor Xa. Therefore, the new tripeptide derivatives areintended to be used as substrates for quantitatively assaying the saidenzymes, more particularly factor Xa.

BACKGROUND ART

Factor Xa is a proteolytic enzyme which is formed in the bloodcoagulation cascade by activation of the proenzyme factor X and which,together with phospholipid and calcium ions, proteolytically splitsfactor II (prothrombin) at two points of the peptide chain and convertsthe said factor into factor IIa (thrombin) which finally causescoagulation. In certain pathological disorders, e.g. liver diseases,vitamin deficiency, etc., and in the dicoumarol therapy, the formationof factor X is reduced. In hereditary disturbances in the synthesis offactor X the formation of factor Xa, of course, also reducedcorrespondingly. Therefore, it is important to have at one's disposal adirect enzymatic assay method which allows factor Xa to be assayedphotometrically in blood plasma in a simple and accurate manner.

The main methods for assaying factor Xa are the following:

(a) Biological assay method [cf. "Thrombosis and Bleeding Disorders,"Nils U. Bang, Georg Thieme Verlag, p. 196 (1971)]: Factor X is activatedto factor Xa by means of venom of Russel viper and calcium ions. In aone-step operation prothrombin is activated to thrombin by factor Xa inthe presence of factor V and phospholipids, and thrombin convertsindicator fibrinogen into fibrin. The clotting time is measured. Therequired factors II and V and fibrinogen are supplied by a substratewhich is free from factor X. The clotting time is influenced by thedegree of activation of factor X. The activation degree, under otherwiseconstant conditions, is a function of the concentration of factor X inthe sample. This biological method allows no more than a rough assay tobe carried out since the clotting time is read off subjectively by theexperimentator. Furthermore, manipulated plasma is required during thepreparation of which mistakes can occur. Moreover, the fibrinogen whichis acting as an indicator is not formed directly, but via activatedthrombin (indirect method).

(b) Biochemical method [cf. "Thrombosis and Bleeding Disorders," N. U.Bang, Georg Thieme Verlag, p. 196/7 (171)]: If the factor X preparationsto be tested are sufficiently pure, a more accurate assay method can beapplied. Esnouf and Williams (1962) have shown that factor Xa has anesterase activity and hence splits synthetic amino acid esters. However,50 to 100 μg of factor X are required for this assay. On the other hand,lower concentrations of factor Xa can be determined by usingcarbobenzoxy-phenylalanine p-nitrophenyl ester and measuring thequantity of p-nitrophenol released per time unit. This assaying methodhas the following disadvantages: The ester used undergoes anautohydrolysis at the applied pH of 8 and, moreover, is not specific forfactor Xa since it responds also to many other enzymes. The ester is notsoluble in water so that acetone is required. As a result of all thesedisadvantages this assaying method is inaccurate and costly.

(c) The published German patent application OS No. 25 52 570 disclosestetrapeptide derivatives which are intended to be used as substrates forassaying factor Xa. Bz-Ile-Glu-Gly-Arg-pNA.HCl is disclosed as anexample of a tetrapeptide derivative which is split by factor Xa withformation of p-nitroaniline. The formation of p-nitroaniline can befollowed spectrophotometrically. This method of assaying factor Xa issomewhat more accurate than the above-described biological andbiochemical assaying methods.

However, the tetrapeptide derivatives described in German patentapplication DE-OS No. 25 52 570 are not sufficiently soluble in aqueousmedia to allow the assay of factor Xa to be carried out at substratesaturation. In the case where extremely low concentrations of factor Xahave to be determined, e.g. in pathological plasma, the saidtetrapeptide derivatives are not sufficiently sensitive to allowreasonably accurate measuring values to be obtained. If the quantity offactor Xa to be measured were increased by adding a further quantity ofplasma, a precipitation of the tetrapeptide substrate would take placeunder the influence of plasmaproteins, and as a result it would beimpossible to perform the enzyme assay.

DISCLOSURE OF INVENTION

Now, new tripeptide derivatives were found which are very easily solublein aqueous media and which have a surprisingly high sensitivity tofactor Xa.

The tripeptide derivatives of the invention have the following generalformula ##STR2## wherein R¹ represents an alkanoyl group which has 2 to8 carbon atoms and which may carry an amino group in the ω-position, aphenylalkanoyl group which has 2 to 4 carbon atoms in the alkanoylradical and the phenyl radical of which may be substituted with an aminogroup in the p-position, a cyclohexylcarbonyl group which may besubstituted with an aminomethyl radical in the 4-position, a benzoylgroup which may be substituted with methyl, amino or halogen, e.g. Cl orBr, in the o- or p-position, an alkoxycarbonyl group having 1 to 8carbon atoms in the alkoxy group, a benzyloxycarbonyl group which may besubstituted with methoxy, methyl or chlorine in the p-position, analkanesulfonyl group having 1 to 4 carbon atoms, a phenylsulfonyl groupwhich may be methylated in the p-position or an α- or β-naphthylsulfonylgroup,

R² represents a straight-chained or branched alkyl radical having 1 to6, preferably 1 to 4, carbon atoms, a hydroxyalkyl radical having 1 to 2carbon atoms, an alkoxyalkyl radical having 1 to 2 carbon atoms in thealkyl and 1 to 4 carbon atoms in the alkoxy, a benzyloxyalkyl radicalhaving 1 to 2 carbon atoms in the alkyl, an ω-carboxyalkyl orω-alkoxycarbonylalkyl radical which has 1 to 3 carbon atoms in the alkyland the alkoxy group of which is straight-chained or branched and has 1to 4 carbon atoms, an ω-benzyloxycarbonylalkyl radical having 1 to 3carbon atoms in the alkyl, or a cyclohexyl-, cyclohexylmethyl-,4-hydroxycyclohexylmethylphenyl-, benzyl-, 4-hydroxybenzyl- orimidazol-4-yl-methyl radical,

R³ represents hydrogen or a straight-chained or branched alkyl radicalhaving 1 to 4 carbon atoms,

R⁴ represents hydrogen or a methyl or ethyl radical, and

R⁵ represents an amino group which is substituted with aromatic orheterocyclic radicals and which is capable of being split offhydrolytically with formation of a coloured or fluorescent compoundH-R⁵.

Preferably, the strongly basic guanidine group of arginine isstabilized, e.g. by protonation with a mineral acid such as HCl, HBr, H₂SO₄ or H₃ PO₄, or an organic acid such as formic, acetic, propionic,lactic, citric, oxalic, tartaric, benzoic, phthalic, trichloroacetic ortrifluoroacetic acid. The nature of protonation has no influencewhatsoever on the sensitivity (susceptibility) of the tripeptidesubstrate to the enzymes.

The removable chromogenic group represented by R⁵ in formula I can be,e.g., a p-nitrophenylamino, 1-carboxy-2-nitrophen-5-yl-amino,1-sulfo-2-nitrophen-5-yl-amino, β-naphthylamino,4-methoxy-β-naphthylamino, 5-nitro-α-naphthylamino, quinon-5-yl-amino,8-nitro-quinon-5-yl-amino, 4-methyl-coumar-7-yl-amino or1,3-di(methoxycarbonyl)-phen-5-yl-amino group (derived from5-amino-isophthalic acid dimethyl ester).

The tripeptide derivatives of formula I can be prepared according tomethods known per se which are described hereinafter:

(1) The chromogenic group R⁵ is attached to the carboxy group of theC-terminal arginine, whilst its α-amino group is protected by aprotective group, e.g. a carbobenzoxy or tert.-butoxycarbonyl group, andthe δ-guanidyl group of arginine is protected by protonation, e.g. withHCl, nitration or tosylation. The C-terminal group R⁵ -serves also as aprotective group during the stepwise building up of the peptide chain.The remaining protective groups can be removed selectively as needed inorder to attach the next amino acid derivatives until the desiredpeptide chain is completely built up. Finally, the remaining protectivegroups can be entirely removed without group R⁵ -being affected (cf.e.g. Miklos Bodansky et al., "Peptide Synthesis," IntersciencePublishers, p. 163-165, 1966).

(2) First of all, the peptide chain is built up (according to Bodansky,loc. cit.) whilst the C-terminal carboxyl group of arginine is protectedby a usual ester group, e.g. a methoxy, ethoxy or benzyloxy group. Theester groups can be removed by alkaline hydrolysis, except for thetert.-butoxy group which has to be removed selectively by means oftrifluoroacetic acid. If the δ-guanidyl group of arginine is protonated,the said ester group is removed by trypsin, no racemization taking placein this case. Thereafter, the chromogenic group R⁵ -is introduced. Ifthe δ-guanidino group of arginine is protected by a nitro or tosyl groupand the N-terminal α-amino group of the tripeptide derivative isprotected by a carbobenzoxy group or a p-methyl, p-methoxy orp-chloro-benzyloxycarbonyl group, or a tert.-butoxy group, all theseprotective groups are removed simultaneously. The removal can beachieved by treating the protected tripeptide derivative with anhydrousHF at room temperature, and as a result all the above-mentioned aminoand δ-guanidino protective groups are removed. The removal can also becarried out by treatment with 2 N HBr in glacial acetic acid at roomtemperature if the protected tripeptide derivative does not contain anynitro or tosyl as protective groups.

The tripeptide derivatives of formula I are substantially more readilysoluble in water than the tetrapeptide derivatives described in Germanpatent application DE-OS No. 25 52 570 and thus allow the enzyme assaysto be carried out at substrate saturation required for obtainingreliable measuring results. Besides, the tripeptide substrates offormula I are significantly more sensitive than the tetrapeptidederivatives disclosed in German patent application DE-OS No. 25 52 570and can, therefore, also be used for assaying extremely lowconcentrations of factor Xa.

The published Dutch patent application OS No. 76 07 433 disclosestripeptide derivatives having the general formula H--D--A₁ --A₂ --A₃--NH--R (A₁, A₂, A₃ =amino acid radicals) which have a high sensitivityto enzymes of enzyme class E.C. 3.4.21., e.g. thrombin and plasmin. Thishigh sensitivity is attributed to the fact that the N-terminal D-aminoacid has an unsubstituted α-amino group. Indeed, if the α-amino group ofthe N-terminal D-amino acid is substituted, i.e. if the hydrogen in theformula is replaced by a protective or blocking group, e.g. by benzoylor carbobenzoxy, the sensitivity of the tripeptide derivative to enzymesof enzyme class 3.4.21. is drastically reduced. On the grounds of thesefindings one could have expected that the new tripeptide derivatives offormula I wherein the α-amino group of the N-terminal D-amino acidcarries a protective or blocking group would not or only slightly besplit by enzymes of enzyme class 3.4.21. However, contrary to allexpectations, the new tripeptide substrates surprisingly have a veryhigh sensitivity to factor Xa.

A further advantage of the tripeptide substrates of the invention ascompared to the tetrapeptide derivatives disclosed in German patentapplication DE-OS No. 25 52 570 resides in the fact that their synthesisis simpler and less costly.

The tripeptide substrates of the invention can be used forquantitatively assaying certain enzymes of enzyme class E.C. 3.4.21.(cf. "Enzyme Nomenclature", Elsevier Scientific Publishing Company,Amsterdam, 1973, p. 238 ff.), e.g. factor Xa in blood plasma, and viafactor Xa also for the quantitative assay of other biologicallyimportant factors, e.g. factor X (proenzyme of factor Xa), antifactor Xa(=antithrombin III or heparin cofactor) or heparin in blood plasma.

These various assays can be carried out in the manner describedhereinafter:

(1) Assay of factor Xa and factor X:

Citrated human plasma is used and first activated with Russel vipervenom (RVV=Russel viper venom) in order to convert factor X present inthe plasma into factor Xa. The following reagents are used:

Buffer--TRIS-imidazole buffer, pH 8.4, ionic strength 0.3;

RVV--Freeze-dried Russel viper venom preparation (furnished byLaboratoire Stago, 92600 Asnieres s/Seine, France), dissolved in 2 ml ofbuffer containing CaCl₂ at a concentration of 0.015 M

Substrate--Cbo-D-Leu-Sar-Arg-pNA.AcOH, dissolved in distilled H₂ O,concentration: 2×10⁻³ M.

A test cuvette is charged with 0.2 ml of RVV reagent heated to 37° C.and then with 0.02 ml of human citrated plasma. The two components areimmediately mixed and then incubated for 60 seconds at 37° C. Theresulting activation mixture is diluted with 1.6 ml of buffer heated to37° C. and mixed with 0.2 ml of 2×10⁻³ M substrate solution. Then, thechange in the optical density of the mixture caused by the releasedp-nitroaniline is measured spectrophotometrically at a wave length of405 nm. The measured increase in the optical density per minute isdirectly proportional to the quantity of factor Xa present in theactivated plasma. The measured increase in the optical density perminute is also a measure for the quantity of factor X present in thestarting plasma since as a result of the activation a given quantity offactor X is converted stoichiometrically into the corresponding quantityof factor Xa.

(2) Assay of antifactor Xa:

A test sample is prepared by diluting citrated human plasma withTRIS-imidazole buffer at a ratio of 1:10. 100 μl of a heparin solutionin TRIS-imidazole buffer (containing 3 USP units of heparin per ml ofbuffer) are added to 100 μl of the diluted plasma, and the mixture isincubated for 2 minutes at 37° C. 100 μl of a solution of factor Xapreparation "Diagen" (furnisher: Diagnostic Reagents, Thame, GreatBritain) containing 8.5 units of factor Xa per ml are added to theincubate. The mixture is incubated for 3 minutes at 37° C. The incubateis diluted with 0.6 ml of TRIS-imidazole buffer heated to 37° C. andimmediately mixed with 100 μl of a 2×10⁻³ M solution ofCbo-D-Leu-Sar-Arg-pNA.AcOH (substrate) in distilled water heated to 37°C.

In a parallel experiment a blank test sample is prepared in the samemanner, but using the same volumetric quantity of buffer instead ofdiluted plasma.

The increase in the optical density at 405 nm is measuredspectrophotometrically for both samples (test sample and blank sample).The difference between the increase in the optical density of the blanksample per minute (ΔOD_(blank) /min.) and the increase in the opticaldensity of the test sample per min. (ΔOD_(test) /min.) is a measure forthe quantity of factor Xa inhibited by the antifactor Xa-heparin complexand hence a measure for the quantity of antifactor Xa (=antithrombinIII) present in the plasma.

(3) Assay of heparin in blood plasma:

A test sample is prepared by diluting plasma of a heparin-treatedpatient with TRIS-imidazole buffer at a ratio of 1:10. The dilutedplasma is incubated for 1 to 2 minutes at 37° C. 100 μl of a solution offactor Xa preparation "Diagen" (furnisher: Diagnostic Reagents, Thame,Great Britain) containing 8.5 units of factor Xa per ml are added to 200μl of the incubate, and the mixture is incubated for 3 minutes at 37° C.The incubate is diluted with 0.6 ml of TRIS-imidazole buffer heated to37° C. and immediatly mixed with 100 μl of a 2×10⁻³ M solution ofCbo-D-Leu-Sar-Arg-pNA.AcOH (substrate) in distilled water.

In a parallel experiment a blank test sample is prepared in the samemanner, but using the same quantity of correspondingly diluted normalplasma (heparin-free) instead of the diluted heparinized plasma.

The increase in the optical density per minute at 405 nm is measuredspectrophotometrically for both samples. The difference between theincrease in the optical density of the blank sample per minute(ΔOD_(blank) /min.) and the increase in the optical density of the testsample per minute (ΔOD_(test) /min.) is a measure for the inhibitoryactivity of the heparin bound to heparin-cofactor (antithrombin III).

Antifactor Xa (=heparin-cofactor or antithrombin III) alone inhibitsfactor Xa but slowly so that only small quantities of factor Xa areinhibited within a short incubation period (e.g. 3 minutes). However, ifantifactor Xa enters into contact with heparin, the two components forma complex which is a rapid inhibitor of factor Xa and which willcompletely combine with factor Xa within a short incubation time (e.g. 3minutes).

Since the inhibition of factor Xa is proportional to the quantity ofheparin present, as long as sufficient antifactor Xa is present, thequantity of heparin present in the patient's plasma can be determined bymeans of a calibration curve established with normal plasma andincreasing quantities of added heparin on the grounds of thepotentiating action of the added heparin.

In the measurement of the quantity of the coloured split product H-R⁵(p-nitroaniline) released in the reaction between factor Xa and thetripeptide derivatives (substrates) of the invention advantage is takenof the fact that the split product has an UV spectrum which differs fromthat of the substrate and is shifted towards higher wave lengths. Thesubstrates of the invention have an absorption maximum at about 310 nmand a molar extinction coefficient of about 13,000. The absorption ofthe substrate at 405 nm is virtually nil. The split product H-R⁵, i.e.p-nitroaniline, formed by enzymatic hydrolysis of the substrate has anabsorption maximum at 380 nm and a molar extinction coefficient of about13,200. At 405 nm the extinction coefficient is only moderately reduced,i.e. to about 10,400.

In the case of substrates which contain a β-naphthylamino,4-methoxy-β-naphthylamino, coumar-7-yl-amino or isophthylamino group asthe chromogenic group, the quantity of split product H-R⁵ released byfactor Xa is measured by fluorescence-spectrophotometry. In a testsystem comprising factor Xa, buffer and substrate the energy-pooreremitted light is continuously measured at 400 to 470 nm after thefluorescent split product has been continuously excited by energy-richerlight at 300 to 400 nm. The quantity of split product formed per timeunit is a measure for the existing factor Xa activity. According todefinition 1 μmole of split product formed per minute corresponds to 1enzyme unit of factor Xa, as based on a given substrate.

The sensitivity of the above-described assaying methods can be furtherincreased by converting the split product H-R⁵, before measurement ofits quantity, into a more intensely coloured compound by coupling with adiazo compound in the case where R⁵ is a p-nitrophenylamino,1-carboxy-2-nitro-phen-5-yl-amino, 1-sulfo-2-nitro-phen-5-yl-amino,5-nitro-α-naphthylamino or 8-nitro-quinon-5-yl-amino group.

Best Mode For Carrying Out the Invention

In the following working examples the preparation of the tripeptidederivatives of the invention is described in a detailed manner.Temperatures are indicated in centigrades.

The analyses of the eluates and products obtained according to theexamples were carried out by thin layer chromatography using glassplates coated with silicon dioxide gel (Merck, F 254). The thin layerchromatograms were developed by means of the following solvent system:n-butanol/acetic acid/water (3:1:1).

The following abbreviations are used:

D-Aadi=D-α-amino-adipic acid

Ac=acetyl

Ac₂ O=acetic anhydride

AcOH=acetic acid

Ala=L-alanine

D-Ala=D-alanine

AOA=D-α-amino-octanoic acid

Arg=L-arginine

D-Asp=D-aspartic acid

BOC=tert.-butoxycarbonyl

Bu=butyl

But=L-2-aminobutyric acid

D-But=D-2-aminobutyric acid

Bz=benzoyl

Bzl=benzyl

Bz₂ O=benzoic anhydride

ChA=quinonyl amide

D-CHA=D-3-cylohexylalanine

D-CHG=D-2-cyclohexylglycine

D-CHT=D-3-(4-hydroxycyclohexyl)-alanine=tyrosine substituted in thenucleus

Cbo=carbobenzoxy

DMF=dimethylformamide

DPA=dimethyl ester of 5-amido-isophthalic acid

TLC=thin layer chromatography or thin layer chromatogram

Et=ethyl

Et₃ N=triethylamine

Gly=glycine

D-Glu=D-glutamic acid

D-His=D-histidine

HMPTA=N,N,N',N',N",N"-hexylmethyl-phosphoric acid triamide

D-Ile=D-isoleucine

D-Leu=D-leucine

SS=solvent system

MCA=7-amido-4-methylcoumarin

MeO=methoxy

MeOH=methanol

NA=naphthylamide

D-Nleu=D-norleucine

D-Nval=D-norvaline

OtBu=tert.-butoxy

OpNP=p-nitrophenoxy

pNA=p-nitroanilide

Pr=propyl

D-Ph'Gly=D-2-phenylglycine

D-Phe=D-phenylalanine

Sar=sarkosine=N-methylglycine

D-Ser=D-serine

TFA=trifluoroacetic acid

THF=tetrahydrofuran

D-Thr=D-threonine

Tos=p-toluenesulfonyl

D-Tyr=D-tyrosine

D-Val=D-valine

If not otherwise stated, the amino acids have the L-form.

EXAMPLE 1 Cbo-D-Leu-Gly-Arg-pNA.HBr

1a. Cbo-Arg-pNA.HCl

In a 250 ml three-necked flask 16.0 g (47 mM) of Cbo-Arg-OH.HCl, whichhad been dried in vacuo over P₂ O₅, were dissolved in 90 ml of abs.HMPTA at 20° in the absence of humidity. To the resulting solution therewas added at room temperature first a solution of 4.74 g (47.0 mM) ofEt₃ N in 10 ml of HMPTA and then portionwise 16.4 g (100 mM) ofp-nitrophenyl isocyanate (100% excess). After a reaction time of 24hours at 20° the major portion of HMPTA was removed by distillation invacuo. The residue was extracted several times with 30% AcOH. Theresidue was discarded. The combined AcOH extracts were further purifiedby passing them through a column of "Sephadex G-15" equilibrated with30% AcOH and eluted with 30% AcOH. The fraction of the AcOH eluate whichwas split by treatment with trypsin with release of p-nitroaniline wasfreeze-dried. There were thus obtained 12.6 g of an amorphous powderwhich was homogeneous in the SS as shown by TLC. Elementary analysis andcalculation from the empirical formula C₂₀ H₂₅ N₆ O₅ Cl gave thefollowing values: C=51.29% (51.67%), H=5.48% (5.42%), N=17.92% (18.08%),Cl=7.50% (7.63%). The values within brackets have been calculated.

1b. 2HBr.H-Art-pNA

4.65 g (10 mM) of compound 1a were treated, while stirring, with 40 mlof 2 N HBr in glacial acetic acid for 45 min. at 20° in the absence ofmoisture. The amino acid derivative dissolved with CO₂ evolution. Thereaction solution was added dropwise with vigorous stirring to 250 ml ofabsolute ether which resulted in the precipitation of 2HBr.H-Arg-pNA.The ethereal phase was sucked off, whereupon the solid phase was washed4 times with portions of 100 ml of abs. ether in order to substantiallyremove benzyl bromide which had formed as a by-product as well as excessHBr and AcOH. The residue was dissolved in 50 ml of MeOH, the pH wasadjusted to 4.5 by the addition of Et₃ N, and the solution wasconcentrated to dryness in vacuo at 30°. The resulting product wasdissolved in 75 ml of MeOH and passed through a column of "Sephadex"LH-20 (cross-linked dextran gel) equilibrated with MeOH. From a fractionof the eluate there were obtained 4.18 g (91.6% of the theory) ofamorphous compound 1b which was homogeneous in the SS as shown by TLC.Elementary analysis and calculation from the empirical formula C₁₂ H₂₀N₆ O₃ Br₂ gave the following values: C=31.15% (31.60%), H=4.35% (4.42%),N=18.84% (18.43%) and Br=34.81% (35.03%).

1c. Cbo-Gly-Arg-pNA.HBr

4.56 g (10 mM) of compound 1b were dissolved in 30 ml of freshlydistilled DMF, and the solution was cooled to -10°; 1.40 ml (10 mM) ofEt₃ N were added to the solution, while stirring. The formed Et₃ N.HBrwas removed by filtration and washed with a small quantity of cold DMF.3.65 g (11 mM) of Cbo-Gly-OpNP were added at -10° to the filtrate; whilestirring, and the reaction was allowed to proceed for 2-3 hours in theabsence of moisture, whereby the temperature of the reaction solutiongradually mounted to about 20°. The solution was again cooled to -10°and buffered with 0.70 ml (5 mM) of Et₃ N. The reaction solution wasallowed to react for about 2 hours at -10° and for three hours at roomtemperature. This procedure was repeated with 0.70 ml of Et₃ N, andafter 16 hours the reaction solution was concentrated to dryness invacuo at 50°. The residue was dissolved in 75 ml of 50% AcOH andpurified by gel filtration on a column of "Sephadex" G-15 equilibratedwith 50% AcOH. The fraction of the AcOH eluate which was split bytreatment with trypsin with release of p-nitroaniline was concentratedto dryness in vacuo at 40°. The residue was dissolved in 150 ml of MeOHand again concentrated to dryness. The resulting residue was dried in avacuum desiccator at 60° over P₂ O₅ to obtain 5.85 g (88.3% of thetheory) of amorphous compound 1c which was homogeneous in the SS asshown by TLC. Elementary analysis and calculation from the empiricalformula C₂₂ H₂₈ N₇ O₆ Br gave the following values: C=46.33% (46.65%),H=5.04% (4.98%), N=17.88% (17.31%) and Br=14.20% (14.11%).

1d. 2HBr.H-Gly-Arg-pNA

4.56 g (8 mM) of compound 1c were treated, while stirring, with 32 ml of2 N HBr in glacial acetic acid for 40 min. at 20°. The dipeptidederivative gradually dissolved with CO₂ evolution. The reaction solutionwas added dropwise with vigorous stirring to 250 ml of abs. ether, andthis resulted in the precipitation of 2HBr.-H-Gly-Arg-pNA. The etherealphase was sucked off, whereupon the solid phase was washed 4 times withportions of 100 ml of abs. ether in order to substantially remove benzylbromide which had formed as a by-product as well as excess HBr and AcOH.The residue was dissolved in 50 ml of MeOH. The pH was adjusted to 4.5by means of Et₃ N, and the solution was concentrated to dryness in vacuoat 30°. The resulting residue was dissolved in 50 ml of MeOH andpurified on a column of "Sephadex" LH-20 equilibrated with MeOH. Thefraction of the MeOH eluate which was split by treatment with trypsinwith release of p-nitroaniline was concentrated to dryness in vacuo at30°. The resulting residue was dried in a vacuum desiccator at 40° overP₂ O₅ to obtain 3.78 g (92.1% of the theory) of amorphous compound 1dwhich was homogeneous in the SS as shown by TLC. Elementary analysis andcalculation from the empirical formula C₁₄ H₂₃ N₇ O₄ Br₂ gave thefollowing values: C=32.31% (32.77%), H=4.59% (4.52%), N=19.47% (19.11%)and Br=30.78% (31.14%).

1e. Cbo-D-Leu-Gly-Arg-pNA.HBr

2.57 g (5 mM) of compound 1d were dissolved in 20 ml of freshlydistilled DMF, and the solution was cooled to -10°; 0.70 ml (5 mM) ofEt₃ N were added to the solution, while stirring. The formed Et₃ N.HBrwas removed by filtration and washed with a small quantity of cold DMF.2.13 g (5.5 mM) of Cbo-D-Leu-OpNP were added at -10° to the filtrate,while stirring. The reaction mixture was allowed to react for 2-3 hoursin the absence of moisture, whereby the temperature of the reactionsolution gradually mounted to about 20°. The solution was again cooledto -10° and buffered with 0.35 ml (2.5 mM) of Et₃ N. The reactionsolution was allowed to react for about 2 hours at -20° and for afurther 3 hours at room temperature. This procedure was repeated with0.35 ml of Et₃ N, and after 16 hours the reaction solution wasconcentrated to dryness in vacuo at 50°. The residue ws dissolved in 50ml of 50% AcOH and purified by gel filtration on a column of "Sephadex"G-15 equilibrated with 50% AcOH. The fraction of the AcOH eluate whichwas split by treatment with trypsin with release of p-nitroaniline wasconcentrated to dryness in vacuo at 40°. The residue was dissolved in100 ml of MeOH, and the solution was again concentrated to dryness. Theresulting residue was dried in a vacuum desiccator at 60° over P₂ O₅ toobtain 3.08 g (90.6% of the theory) of amorphous compound 1e which washomogeneous in the SS as shown by TLC. Elementary analysis andcalculation from the empirical formula C₂₈ H₃₉ N₈ O₇ Br gave thefollowing values: C=49.06% (49.49%), H=5.82% (5.78%), N=16.85% (16.49%)and Br=11.59% (11.76%).

The amino acid analysis confirmed the presence of the expected aminoacids in the correct proportions: Gly:1.00--D-Leu:0.99--Arg:0.97.

EXAMPLE 2 Cbo-D-Leu-Gly-Arg-MCA.HBr

2b. 2HBr.H-Arg-MCA

13.0 g (25.9 mM) of commercial Cbo-Arg-MCA.HCl were deblocked accordingto Example 1b by means of 104 ml (208 mM) of a solution of 2 N HBr inglacial acetic acid. The dry residue was dissolved in 400 ml of MeOH andpurified on a column of "Sephadex" LH-20. The fraction of the MeOHeluate which was split by treatment with trypsin with release of4-methyl-7-amino-coumarin was concentrated to dryness in vacuo at 30°.The resulting residue was dried in a vacuum desiccator at 40° over P₂ O₅to obtain 11.2 g (87.7% of the theory) of amorphous compound 2b whichwas homogeneous in the SS as shown by TLC. Elementary analysis andcalculation from the empirical formula C₁₆ H₂₃ N₅ O₃ Br₂ gave thefollowing values: C=39.40% (38.96%), H=4.61% (4.70%), N=14.48% (14.20%)and Br=31.90% (32.40%).

2c. Cbo-Gly-Arg-MCA.HBr

4.93 g (10 mM) of compound 2b and 3.65 g (11 mM) of Cbo-Gly-OpNP wereadded to 75 ml of freshly distilled DMF. After cooling to -10°, therewere added, while stirring, first 1.40 ml (10 mM) and then 0.70 ml (5mM) of Et₃ N. The mixture was allowed to react, in the absence ofhumidity, first for 3 hours at -10° and then for 4 hours at roomtemperature. The reaction solution was again cooled to -10°, bufferedwith 0.70 ml of Et₃ N and stirred overnight at 20°. The reaction mixturewas concentrated to dryness in vacuo at 50°, and the residue wasdissolved in 200 ml of 50% AcOH and purified on a column of "Sephadex"G-15. The fraction of the AcOH eluate which was split by treatment withtrypsin with release of 4-methyl-7-amino-coumarin was concentrated todryness in vacuo at 40°. The thus obtained residue was dried in a vacuumdesiccator at 60° over P₂ O₅ to yield 4.98 g (82.5% of the theory) ofamorphous compound 2c which was homogeneous in the SS as shown by TLC.Elementary analysis and calculation from the empirical formula C₂₆ H₃₁N₆ O₆ Br gave the following values: C=51.48% (51.75%), H=5.24% (5.18%),N=13.70% (13.93%) and Br=13.14% (13.24%).

2d. 2HBr.H-Gly-Arg-MCA

4.83 g (8 mM) of compound 2c were deblocked according to Example 1d bymeans of 32 ml of 2 N HBr in glacial acetic acid. The resulting crudeproduct was dissolved in 100 ml of MeOH and purified on a column of"Sephadex" LH-20. The fraction of the MeOH eluate which was split bytreatment with trypsin with release of 4-methyl-7-amino-coumarin wasconcentrated to dryness in vacuo at 30°. The resulting residue was driedin a vacuum desiccator at 40° over P₂ O₅ to give 4.05 g (92.0% of thetheory) of amorphous compound 2d which was homogeneous in the SSaccording to TLC. Elementary analysis and calculation from the empiricalformula C₁₈ H₂₆ N₆ O₄ Br₂ gave the following values: C=39.02% (39.29%),H=4.78% (4.76%), N=15.39% (15.27%) and Br=28.72% (29.04%).

2e. Cbo-D-Leu-Gly-Arg-MCA.HBr

2.75 g (5 mM) of compound 2d were reacted with 2.13 g (5.5 mM) ofCbo-D-Leu-OpNP in accordance with Example 1e. The resulting crudeproduct was dissolved in 75 ml of 50% AcOH and purified on a column of"Sephadex" G-15. The fraction of the AcOH eluate which was split bytreatment with trypsin with release of 4-methyl-7-amino-coumarin wasconcentrated to dryness in vacuo at 40°. The residue was dried in avacuum desiccator at 60° over P₂ O₅ to obtain 2.91 g (81.2% of thetheory) of amorphous compound 2e which was homogeneous in the SS asshown by TLC. Elementary analysis and calculation from the empiricalformula C₃₂ H₄₂ N₇ O₇ Br gave the following values: C=53.13% (53.63%),H=6.01% (5.91%), N=13.91% (13.68%) and Br=10.88% (11.15%).

The amino acid analysis confirmed the presence of the expected aminoacids in the correct proportions: Gly:1.00--Leu:1.02--Arg:0.98.

EXAMPLE 3 Cbo-D-Leu-Gly-Arg-DPA.HBr

3a. Cbo-Arg-DPA.HCl

34.48 g (0.1 mole) of dried Cbo-Arg-OH.HCl were dissolved in a 1000 mlthree-necked flask in a mixture of 150 ml of freshly distilled anhydrousDMF and 300 ml of abs. THF at 20°. To the solution, cooled to -10°,there were added, while stirring, 10.2 g (0.1 mole) of Et₃ N in theabsence of humidity. Then a solution of 13.65 g (0.1 mole) of isobutylchloroformate in 50 ml of THF was added dropwise to the mixture within20 min., whereby the reaction temperature was never allowed to exceed-5°. After an additional reaction time of 10 minutes at a temperature of-10° to -5° a solution of 20.92 g (0.1 mole) of dimethyl5-amino-isophthalate in 75 ml of DMF was added dropwise to the reactionmixture within 30 min., whereby the reaction temperature was always keptbelow -5°. The reaction mixture was allowed to react for another hour at-5°. Then it was stirred overnight at 20° and subsequently cooled to-15° in order to let the Et₃ N.HCl crystallize. The formed Et₃ N.HCl wasfiltered off and washed with a small amount of cold DMF. The filtrateand the washing solution were concentrated to dryness in vacuo at 50°.The residue was dissolved in 1000 ml of 50% AcOH and purified by gelfiltration on a column of "Sephadex" G-15 equilibrated with 50% AcOH.The fraction of the AcOH eluate which was split by treatment withtrypsin with release of dimethyl 5-amino-isophthalate was concentratedto dryness in vacuo at 40°. The residue was dried in a vacuum desiccatorat 50° over P₂ O₅ to obtain 24.6 g (45.9% of the theory) of amorphouscompound 3a which was homogeneous in the SS as shown by TLC. Elementaryanalysis and calculation from the empirical formula C₂₄ H₃₀ N₅ O₇ Clgave the following values: C=53.21% (53.78%), H=5.71% (5.64%), N=13.20%(13.07%) and Cl=6.52% (6.62%).

3b. 2HBr.H-Arg-DPA

21.44 g (40 mM) of compound 3a were deblocked according to Example 1b.After the usual treatment the resulting crude product was dissolved in250 ml of MeOH and purified by gel filtration on a column of "Sephadex"LH-20. The fraction of the MeOH eluate which was split by treatment withtrypsin with release of dimethyl 5-amino-isophthalate was concentratedto dryess in vacuo. The residue was dried in a vacuum desiccator at 40°over P₂ O₅ to obtain 19.63 g (93.1% of the theory) of amorphous compound3b which was homogeneous in the SS as shown by TLC. Elementary analysisand calculation from the empirical formula C₁₆ H₂₅ N₅ O₅ Br₂ gave thefollowing values: C=36.82% (36.45%), H=4.67% (4.78%), N=13.45% (13.28%)and Br=29.85% (30.31%).

3c. Cbo-Gly-Arg-DPA.HBr

5.27 g (10 mM) of compound 3b were reacted according to Example 1c with3.65 g (11 mM) of Cbo-Gly-OpNP. The crude product obtained after theusual treatment was dissolved in 200 ml of 50% AcOH and purified on acolumn of "Sephadex" G-15. The fraction of the AcOH eluate which wassplit by treatment with trypsin with release of dimethyl5-amino-isophthalate was concentrated to dryness in vacuo at 40°. Theresidue was dried in a vacuum desiccator at 60° over P₂ O₅ to give 5.29g (83.0% of the theory) of amorphous compound 3c which was homogeneousin the SS as shown by TLC. Elementary analysis and calculation from theempirical formula C₂₆ H₃₃ N₆ O₈ Br gave the following values: C=48.50%(48.99%), H=5.28% (5.22%), N=12.92% (13.18%) and Br=12.33% (12.53%).

3d. 2HBr.H-Gly-Arg-DPA

5.10 g (8 mM) of compound 3c were deblocked according to Example 1d bymeans of 32 ml of 2 N HBr in glacial acetic acid. The crude productobtained after the usual treatment was dissolved in 100 ml of MeOH andpurified on a column of "Sephadex" LH-20. The fraction of the MeOHeluate which was split by treatment with trypsin with release ofdimethyl 5-amino-isophthalate was concentrated to dryness in vacuo at30°. The residue was dried in a vacuum desiccator at 40° over P₂ O₅ togive 4.25 g (90.9% of the theory) of amorphous compound 3d which washomogeneous in the SS as shown by TLC. Elementary analysis andcalculation from the empirical formula C₁₈ H₂₈ N₆ O₆ Br₂ gave thefollowing values: C=36.85% (37.00%), H=4.90% (4.83%), N=14.72% (14.38%)and Br=26.95% (27.35%).

3e. Cbo-D-Leu-Gly-Arg-DPA-HBr

2.92 g (5 mM) of compound 3d were reacted according to Example 1e with2.13 g (5.5 mM) of Cbo-D-Leu-OpNP. The crude product obtained after theusual treatment was dissolved in 100 ml of 50% AcOH and purified on acolumn of "Sephadex" G-15. The fraction of the AcOH eluate which wassplit by treatment with trypsin with relesase of dimethyl5-amino-isophthalate was concentrated to dryness in vacuo at 40°. Theresidue was dried in a vacuum desiccator at 60° over P₂ O₅ to obtain3.11 g (82.9% of the theory) of amorphous compound 3e which washomogeneous in the SS as shown by TLC. Elementary analysis andcalculation from the empirical formula C₃₂ H₄₄ N₇ O₉ Br gave thefollowing values: C=50.88% (51.20%), H=5.99% (5.91%), N=13.26% (13.06%)and Br=10.48% (10.64%).

The amino acid analysis confirmed the presence of the expected aminoacids in the correct proportions: Gly:1.00--Leu:1.00--Arg:0.97.

EXAMPLE 4 Cbo-D-Leu-Sar-Arg-2-NA.HBr

4b. 2HBr.H-Arg-2-NA

9.40 g (20 mM) of commercial Cbo-Arg-2-NA.HCl were deblocked accordingto Example 1b with a solution of 80 ml of 2 N HBr in glacial aceticacid. The product obtained after the usual treatment was dissolved in150 ml of MeOH and purified on a column of "Sephadex" LH-20. Thefraction of the MeOH eluate which was split by treatment with trypsinwith release of 2-naphthylamine was concentrated to dryness in vacuo at30°. The residue was dried in a vacuum desiccator at 40° over P₂ O₅ toobtain 8.60 g (93.2% of the theory) of amorphous compound 4b which washomogeneous in the SS as shown by TLC. Elementary analysis andcalculation from the empirical formula C₁₆ H₂₃ N₅ OBr₂ gave thefollowing values: C=42.08% (41.67%), H=5.12% (5.03%), N=14.68% (15.19%)and Br=33.96% (34.65%).

4c. Cbo-Sar-Arg-2-NA.HBr

4.6 g (10 mM) of compound 4b were reacted according to Example 1c with3.80 g (11 mM) of Cbo-Sar-OpNP. The crude product obtained after theusual treatment with dissolved in 150 ml of 50% AcOH and purified on acolumn of "Sephadex" G-15. The fraction of the AcOH eluate which wassplit by treatment with trypsin with release of 2-naphthylamine wasconcentrated to dryness in vacuo at 40°. The residue was dried in avacuum desiccator at 60° over P₂ O₅ to obtain 4.95 g (84.5% of thetheory) of amorphous compound 4c which was homogeneous in the SS asshown by TLC. Elementary analysis and calculation from the empiricalformula C₂₇ H₃₃ N₆ O₄ Br gave the following values: C=55.72% (55.39%),H=6.73% (5.68%), N=14.68% (14.35%) and Br=13.42% (13.65%).

4d. 2HBr.H-Sar-Arg-2-NA

4.68 g (8 mM) of compound 4c were deblocked according to Example 1d bymeans of 28 ml of 2 N HBr in glacial acetic acid. The crude productobtained after the usual treatment was dissolved in 100 ml of MeOH andpurified on a column of "Sephadex" LH-20. The fraction of the MeOHeluate which was split by treatment with trypsin with release of2-naphthylamine was concentrated to dryness in vacuo at 30°. The residuewas dried in a vacuum desiccator at 40° over P₂ O₅ to give 4.08 g (95.8%of the theory) of amorphous compound 4d which was homogeneous in the SSas shown by TLC. Elementary analysis and calculation from the empiricalformula C₁₉ H₂₈ N₆ O₂ Br₂ gave the following values: C=43.9% (42.87%),H=5.32% (5.30%), N=16.02% (15.79%) and Br=29.68% (30.02%).

4e. Cbo-D-Leu-Sar-Arg-2-NA.HBr

2.66 g (5 mM) of compound 4d were reacted according to Example 1e with2.13 g (5.5 mM) of Cbo-D-Leu-OpNP. The crude product obtained after theusual treatment was dissolved in 100 ml of 50% AcOH and purified on acolumn of "Sephadex G-15". The first main fraction of the AcOH eluatewhich was split by treatment with trypsin with release of2-naphthylamine was concentrated to dryness in vacuo at 40° and dried ina vacuum desiccator at 60° over P₂ O₅. There were thus obtained 3.01 g(86.2% of the theory) of amorphous compound 4e which was homogeneous inthe SS as shown by TLC. Elementary analysis and calculation from theempirical formula C₃₃ H₄₄ N₇ O₅ Br gave the following values: C=57.05%(56.73%), H=6.40% (6.35%), N=14.30% (14.03%) and Br=11.12% (11.44%).

The amino acid analysis confirmed the presence of the expected aminoacids in the correct proportions: Sar:1.00--Leu:1.02--Arg:0.97.

EXAMPLE 5 Cbo-D-Leu-Sar-Arg-4-MeO-2-NA.HBr

5b. 2HBr.H-Arg-4-MeO-2-NA

10.0 g (20 mM) of commercial Cbo-Arg-4-MeO-2-NA.HCl were deblockedaccording to Example 1b by means of 80 ml of 2 N HBr in glacial aceticacid. The crude product obtained after the usual treatment was dissolvedin 150 ml of MeOH and purified on a column of "Sephadex" LH-20. The mainfraction of the MeOH eluate which was split by treatment with trypsinwith release of 4-methoxy-2-naphthylamine was concentrated to dryness invacuo at 30°. The residue was dried in a vacuum desiccator at 40° overP₂ O₅ to give 8.98 g (91.4% of the theory) of amorphous compound 5bwhich was homogeneous in the SS as shown by TLC. Elementary analysis andcalculation from the empirical formula C₁₇ H₂₅ N₅ O₂ Br₂ gave thefollowing values: C=41.22% (41.57%), H=5.19% (5.13%), N=14.40% (14.26%)and Br=32.01% (32.53%).

5c. Cbo-Sar-Arg-4-MeO-2-NA.HBr

4.91 g (10 mM) of compound 5b were reacted according to Example 1c with3.80 g (11 mM) of Cbo-Sar-OpNP. The crude product obtained after theusual treatment was dissolved in 150 ml of 50% AcOH and purified on acolumn of "Sephadex" G-15. The first main fraction of the AcOH eluatewhich was split by treatment with trypsin with release of4-methoxy-2-naphthylamine was concentrated to dryness in vacuo at 40°.The residue was dried in a vacuum desiccator at 60° over P₂ O₅ to obtain4.86 g (79.0% of the theory) of amorphous comound 5c which washomogeneous in the SS as shown by TLC. Elementary analysis andcalculation from the empirical formula C₂₈ H₃₅ N₆ O₅ Br gave thefollowing values: C=54.38% (54.64%), H=5.81% (5.73%), N=13.93% (13.65%)and Br=12.75% (12.98%).

5d. 2HBr.H-Sar-Arg-4-MeO-2-NA

4.31 g (7 mM) of compound 5c were deblocked according to Example 1d with28 ml of 2 N HBr in glacial acetic acid. The crude product obtainedafter the usual treatment was dissolved in 100 ml of MeOH and purifiedon a column of "Sephadex" LH-20. The main fraction of the MeOH eluatewhich was split by treatment with trypsin with formation of4-methoxy-2-naphthylamine was concentrated to dryness in vacuo at 30°.The residue was dried in a vaccum desiccator at 40° over P₂ O₅ to obtain3.74 g (95.0% of the theory) of amorphous compound 5d which washomogeneous in the SS as shown by TLC. Elementary analysis andcalculation from the empirical formula C₂₀ H₃₀ N₆ O₃ Br₂ gave thefollowing values: C=43.01% (42.72%), H=5.44% (5.38%), N=15.25% (14.95%)and Br=28.03% (28.42%).

The amino acid analysis confirmed the presence of the expected aminoacids in the correct proportions: Sar:1.00--Leu:1.01--Arg:0.98.

5e. Cbo-D-Leu-Sar-Arg-4-MeO-2-NA.HBr

2.81 g (5 mM) of compound 5d were reacted according to Example 1e with2.13 g (5.5 mM) of Cbo-D-Leu-OpNP. The crude product obtained after theusual treatment was dissolved in 125 ml of 50% AcOH and purified on acolumn of "Sephadex" G-15. The first main fraction of the AcOH eluatewhich was split by treatment with trypsin with release of4-methoxy-2-naphthylamine was concentrated to dryness in vacuo at 40°.The residue was dried in a vacuum desiccator at 60° over P₂ O₅ to give2.98 g (81.8% of the theory) of amorphous compound 5e which washomogeneous in the SS as shown by TLC. Elementary analysis andcalculation from the empirical formula C₃₄ H₄₆ N₇ O₆ Br gave thefollowing values: C=56.28% (56.04%), H=6.34% (6.36%), N=13.75% (13.46%)and Br=10.68% (10.97%).

EXAMPLE 6 Cbo-D-Leu-Gly-Arg-pNA.AcOH

6.80 g (10 mM) of Cbo-D-Leu-Gly-Arg-pNA.HBr (prepared according toExample 1) were dissolved in 75 ml of 60% aqueous MeOH. The solution waspoured on a column of "Amberlite" JRA-401 in the acetate form. Thecolumn was eluted by means of 60% aqueous MeOH, the HBr being replacedby AcOH by ion exchange. The eluate was concentrated to dryness in vacuoat 40°. After drying in the vacuum desiccator at 40° over P₂ O₅ therewere obtained 6.62 g of bromide-free Cbo-D-Leu-Gly-Arg-pNA.AcOH (99.0%of the theory). Other salts with organic acids, e.g. formic acid,propionic acid, oxalic acid, tartaric acid, citric acid, lactic acid,benzoic acid, chlorobenzoic acid, salicylic acid or phthalic acid, canalso be prepared from the above named tripeptide derivative according tothe same method. The ion exchanger can be e.g. "Amberlite" JRA-401 inits hydrochloride form, and the desired acid salt form can be obtainedby converting the said ion exchanger into the basic OH-form by treatmentwith caustic soda solution and then with a solution of a 1:1 mixture ofthe desired organic acid and its sodium salt in 60% aqueous MeOH.

EXAMPLE 7 Tos-D-Leu-Gly-Arg-pNA.HBr

6.26 g (10 mM) of 2HBr.H-D-Leu-Gly-Arg-pNA (prepared according toExample 7f, see Table 3) were dissolved in 30 ml of distilled DMF. Thesolution was cooled to -15°, and first 2.80 ml (20 mM) of Et₃ N andimmediately afterwards 1.91 g (10 mM) of tosyl chloride were added,while stirring, in the absence of humidity. After a reaction time ofabout 2 to 3 hours at -15°, the reaction mixture was allowed to furtherreact overnight at room temperature. The reaction mixture was againcooled to -15°. The precipitated mixture of Et₃ N.HBr and Et₃ N.HCl wasremoved by filtration and washed with a small amount of cold DMF. Thefiltrate and the washing solution were combined and concentrated todryness in vacuo at 50°. The residue was dissolved in 75 ml of 50% AcOH.The solution was purified on a column of "Sephadex" G-15 equilibratedwith 50% AcOH. The main fraction of the AcOH eluate which was split bytreatment with trypsin with release of p-nitroaniline was concentratedto dryness in vacuo at 40°. The residue was dissolved in 100 ml of MeOH,and the solution was again concentrated to dryness. The residue wasdried in a vacuum desiccator at 60° over P₂ O₅ to obtain 6.65 g (95.0%of the theory) of amorphous compound Tos-D-Leu-Gly-Arg-pNA.HBr which washomogeneous in the SS as shown by TLC. Elementary analysis andcalculation from the empirical formula C₂₇ H₃₉ N₈ SO₇ Br gave thefollowing values: C=46.85% (46.35%), H=5.66% (5.62%), N=16.28% (16.02%),S=4.43% (4.58%) and Br=11.22% (11.42%).

Instead of using tosyl chloride as the acylation agent correspondingamounts of acetyl chloride, butyryl chloride, octanoyl chloride, benzoicacid chloride, p-methylbenzoic acid chloride, 2-chlorobenzoic acidchloride, methanesulfonyl chloride, n-butanesulfonyl chloride,benzenesulfonyl chloride, α-naphthalenesulfonyl chloride, methylchloroformate, isobutyl chloroformate, octyl chloroformate, benzoic acidanhydride, acetic acid anhydride, phenylacetic acid-OpNP,phenylpropionic acid-OpNP, cyclohexylcarboxylic acid-OpNP,N-Cbo-4-aminomethylcyclohexylcarboxylic acid-OpNP,N-Cbo-ω-amino-n-butyric acid-OpNP, N-Cbo-ωaminooctanoic acid-OpNP orN-Cbo-4-aminomethylbenzoic acid-OpNP can also be used.

                                      TABLE 1                                     __________________________________________________________________________    Ex-                    Starting    Method                                                                              Elementary                           am-                    products    yield analysis    Amino acid               ple                                                                              Final product       (mMole)     %       found %                                                                            calc.%                                                                             analysis                 __________________________________________________________________________    8  Cbo--D-Nleu--Gly--Arg--pNA.HBr                                                                    1d (5 Mole) Example 1e                                                                          C 48.95                                                                              49.49                                                                              Gly:Nleu:Arg                C.sub.28 H.sub.39 N.sub.8 O.sub.7 Br                                                              Cbo--D-Nleu--OpNP                                                                         82.8  H 5.83 5.78 1.00:0.99:0.97                                  (5.5 mMole)       N 16.80                                                                              16.49                                                                  Br                                                                              11.55                                                                              11.76                         9  Cbo--D-Nval--Gly--Arg--pNA.HBr                                                                    1d (5 mMole)                                                                              Example 1e                                                                          C 48.58                                                                              48.73                                                                              Gly:Nval:Arg                C.sub.27 H.sub.37 N.sub.8 O.sub.7 Br                                                              Cbo--D-Nval--OpNP                                                                         84.2  H 5.66 5.60 1.00:1.02:0.98                                  (5.5 mMole)       N 16.98                                                                              16.84                                                                  Br                                                                              11.86                                                                              12.01                         10 Cbo--D-But--Gly--Arg--pNA.HBr                                                                     1d (5 mMole)                                                                              Example 1e                                                                          C 47.73                                                                              47.93                                                                              Gly:But:Arg                 C.sub.26 H.sub.35 N.sub.8 O.sub.7 Br                                                              Cbo--D-But--OpNP                                                                          83.8  H 5.48 5.41 1.00:0.99:0.97                                  (5.5 mMole)       N 17.48                                                                              17.20                                                                  Br                                                                              12.11                                                                              12.26                         11 Cbo--D-Ala--Gly--Arg--pNA.HBr                                                                     1d (5 mMole)                                                                              Example 1e                                                                          C 46.88                                                                              47.10                                                                              Gly:Ala:Arg                 C.sub.25 H.sub.33 N.sub.8 O.sub.7 Br                                                              Cbo--D-Ala--OpNP                                                                          80.5  H 5.26 5.22 1.00:1.01:0.98                                  (5.5 mMole)       N 17.85                                                                              17.58                                                                  Br                                                                              12.33                                                                              12.53                         12 Cbo--D-CHA--Gly--Arg--pNA.HBr                                                                     1d (5 mMole)                                                                              Example 1e                                                                          C 50.75                                                                              51.07                                                                              Gly:CHA:Arg                 C.sub.30 H.sub.41 N.sub.8 O.sub.7 Br                                                              Cbo--D-CHA--OpNP                                                                          82.7  H 5.93 5.86 1.00:0.98:0.99                                  (5.5 mMole)       N 16.10                                                                              15.88                                                                  Br                                                                              11.18                                                                              11.32                         13 Cbo--D-CHG--Gly--Arg--pNA.HBr                                                                     1d (5 mMole)                                                                              Example 1e                                                                          C 50.18                                                                              50.36                                                                              Gly:CHG:Arg                 C.sub.29 H.sub.39 N.sub.8 O.sub.7 Br                                                              Cbo--D-CHG--OpNP                                                                          83.5  H 5.69 5.68 1.00:0.97:0.99                                  (5.5 mMole)       N 16.38                                                                              16.20                                                                  Br                                                                              11.29                                                                              11.55                         14 Cbo--D-CHT--Gly--Arg--pNA.HBr                                                                     1d (5 mMole)                                                                              Example 1e                                                                          C 49.62                                                                              49.93                                                                              Gly:CHT:Arg                 C.sub.30 H.sub.41 N.sub.8 O.sub.8 Br                                                              Cbo--D-CHT--OpNP                                                                          82.0  H 5.80 5.73 1.00:0.96:1.01                                  (5.5 mMole)       N 15.76                                                                              15.53                                                                  Br                                                                              10.88                                                                              11.07                         15 Cbo--D-Ph'Gly--Gly--Arg--pNA.HBr                                                                  1d (5 mMole)                                                                              Example 1e                                                                          C 50.60                                                                              50.81                                                                              Gly:Ph'Gly:Arg              C.sub.29 H.sub.33 N.sub.8 O.sub.7 Br                                                              Cbo--D-Ph'Gly--                                                                           81.3  H 4.88 4.85 1.00:0.98:0.98                                  OpNP              N 16.63                                                                              16.35                                                (5.5 mMole)       Br                                                                              11.48                                                                              11.66                         16 Cbo--D-Phe--Gly--Arg--pNA.HBr                                                                     1d (5 mMole)                                                                              Example 1e                                                                          C 51.20                                                                              51.51                                                                              Gly:Phe:Arg                 C.sub.30 H.sub.35 N.sub.8 O.sub.7 Br                                                              Cbo--D-Phe--OpNP                                                                          80.0  H 5.07 5.04 1.00:1.01:0.98                                  (5.5 mMole)       N 16.28                                                                              16.02                                                                  Br                                                                              11.35                                                                              11.42                         17 Cbo--D-Val--Gly--Arg--pNA.HBr                                                                     1d (5 mMole)                                                                              Example 1e                                                                          C 48.71                                                                              48.72                                                                              Gly:Val:Arg                 C.sub.27 H.sub.37 N.sub.8 O.sub.7 Br                                                              Cbo--D-Val--OpNP                                                                          82.8  H 5.62 5.60 1.00:1.02:1.00                                  (5.5 mMole)       N 17.08                                                                              16.84                                                                  Br                                                                              11.90                                                                              12.01                         18 Cbo--D-Ile--Gly--Arg--pNA.HBr                                                                     1d (5 mMole)                                                                              Example 1e                                                                          C 49.12                                                                              49.49                                                                              Gly:Ile:Arg                 C.sub.28 H.sub.39 N.sub.8 O.sub.7 Br                                                              Cbo--D-Ile--OpNP                                                                          81.2  H 5.80 5.78 1.00:0.99:0.98                                  (5.5 mMole)       N 16.66                                                                              16.49                                                                  Br                                                                              11.58                                                                              11.76                         19 Cbo--D-Glu(γ-OtBu)--Gly--Arg--                                                              1d (5 mMole)                                                                              Example 1e                                                                          C 49.18                                                                              49.54                                                                              Gly:Glu:Arg                 pNA.HBr             Cbo--D-Glu(γ-OtBu)--                                                                78.2  H 5.82 5.77 1.00:0.96:0.98              C.sub.31 H.sub.43 N.sub.8 O.sub.9 Br                                                              OpNP              N 15.12                                                                              14.91                                                (5.5 mMole)       Br                                                                              10.50                                                                              10.63                         20 Cbo--D-Asp(β-OtBu)--Gly--Arg--                                                               1d (5 mMole)                                                                              Example 1e                                                                          C 48.70                                                                              48.85                                                                              Gly:Asp:Arg                 pNA.HBr             Cbo--D-Asp(β-OtBu)--                                                                 77.6  H 5.66 5.60 1.00:0.97:1.00              C.sub.30 H.sub.41 N.sub.8 O.sub.9 Br                                                              OpNP              N 15.02                                                                              15.19                                                (5.5 mMole)       Br                                                                              10.65                                                                              10.83                         21 Cbo--D-His--Gly--Arg--pNA.2HBr                                                                    1d (5 mMole)                                                                              Example 1e                                                                          C 42.71                                                                              42.87                                                                              Gly:His:Arg                 C.sub.28 H.sub.36 N.sub.10 O.sub.7 Br.sub.2                                                       Cbo--D-His--OpNP                                                                          68.5  H 4.70 4.63 1.00:0.94:0.99                                  (5.5 mMole)       N 18.05                                                                              17.86                                                                  Br                                                                              20.12                                                                              20.37                         22 Cbo--D-Leu--Sar--Arg--pNA.HBr                                                                     22d (5 mMole)                                                                             Example 1e                                                                          C 50.48                                                                              50.22                                                                              Sar:Leu:Arg                 C.sub.29 H.sub.41 N.sub.8 O.sub.7 Br                                                              Cbo--D-Leu--OpNP                                                                          82.2  H 6.02 5.96 1.00:1.01:0.97                                  (5,5 mMole)       N 16.38                                                                              16.16                                                                  Br                                                                              11.33                                                                              11.52                         23 Cbo--D-Leu--Ala--Arg--pNA.HBr                                                                     23d (5 mMole)                                                                             Example 1e                                                                          C 50.48                                                                              50.22                                                                              Ala:Leu:Arg                 C.sub.29 H.sub.41 N.sub.8 O.sub.7 Br                                                              Cbo--D-Leu--OpNP                                                                          83.5  H 6.03 5.96 1.00:1.01:0.98                                  (5.5 mMole)       N 16.28                                                                              16.16                                                                  Br                                                                              11.40                                                                              11.52                         24 CH.sub.3 SO.sub.2 --D-Nleu--Gly--Arg--                                                            36f (3 mMole)                                                                             Examples                                                                            C 45.66                                                                              45.84                                                                              Gly:Nleu:Arg                pNA.AcOH            CH.sub.3 SO.sub.2 --Cl                                                                    7 & 6 H 6.41 6.36 1.00:1.01:0.98              C.sub.23 H.sub.38 N.sub.8 O.sub.9 S                                                               (3.3 mMole) 83.1  N 18.93                                                                              18.59                                                                  S 5.24 5.32                          25 isoButoxy-CO--D-Nleu--Gly--Arg--                                                                  36f (3 mMole)                                                                             Examples                                                                            C 51.63                                                                              51.91                                                                              Gly:Nleu:Arg                pNA.AcOH            isoButoxy-  7 & 6 H 7.12 7.10 1.00:1.02:0.99              C.sub.27 H.sub.44 N.sub.8 O.sub.9                                                                 CO--Cl      81.4  N 18.17                                                                              17.94                                                (3.3 mMole)                                            26 Cbo--D-Leu--N(Et)Gly--Arg--pNA.                                                                   26d (5 mMole)                                                                             Example 1e                                                                          C 51.30                                                                              50.92                                                                              Leu:N(Et)Gly:Arg            HBr                 Cbo--D-Leu--OpNP                                                                          80.6  H 6.15 6.13 1.00:0.98:0.99              C.sub.30 H.sub.43 N.sub.8 O.sub.7 Br                                                              (5.5 mMole)       N 16.10                                                                              15.84                                                                  Br                                                                              11.12                                                                              11.29                         27 Cbo--D-Leu--N(Pr)Gly--Arg--pNA.                                                                   27d (5 mMole)                                                                             Example 1e                                                                          C 51.95                                                                              51.59                                                                              Leu:N(Pr)Gly:Arg            HBr                 Cbo--D-Leu--OpNP                                                                          77.5  H 6.34 6.29 1.00:0.96:0.98              C.sub.31 H.sub.45 N.sub.8 O.sub.7 Br                                                              (5.5 mMole)       N 15.80                                                                              15.53                                                                  Br                                                                              10.83                                                                              11.07                         28 Cbo--D-CHA--Sar--Arg--pNA.HBr                                                                     22d (5 mMole)                                                                             Example 1e                                                                          C 51.48                                                                              51.74                                                                              Sar:CHA:Arg                 C.sub.31 H.sub.43 N.sub.8 O.sub.7 Br                                                              Cbo--D-CHA--OpNP                                                                          84.2  H 6.08 6.02 1.00:0.98:0.98                                  (5.5 mMole)       N 15.76                                                                              15.57                                                                  Br                                                                              10.95                                                                              11.10                         29 Cbo--D-CHT--Sar--Arg--pNA.HBr                                                                     22d (5 mMole)                                                                             Example 1e                                                                          C 50.33                                                                              50.61                                                                              Sar:CHT:Arg                 C.sub.31 H.sub.43 N.sub.8 O.sub.8 Br                                                              Cbo--D-CHT--OpNP                                                                          80.9  H 5.96 5.89 1.00:0.97:0.99                                  (5.5 mMole)       N 15.48                                                                              15.23                                                                  Br                                                                              10.70                                                                              10.86                         30 Cbo--D-Nleu--Sar--Arg--pNA.HBr                                                                    22d (5 mMole)                                                                             Example 1e                                                                          C 50.31                                                                              50.22                                                                              Sar:Nleu:Arg                C.sub.29 H.sub.41 N.sub.8 O.sub.7 Br                                                              Cbo--D-Nleu--OpNP                                                                         80.6  H 6.00 5.96 1.00:1.02:0.98                                  (5.5 mMole)       N 16.40                                                                              16.16                                                                  Br                                                                              11.38                                                                              11.52                         31 Cbo--D-Nval--Sar--Arg--pNA.HBr                                                                    22d (5 mMole)                                                                             Example 1e                                                                          C 49.33                                                                              49.49                                                                              Sar:Nval:Arg                C.sub.28 H.sub.39 N.sub.8 O.sub.7 Br                                                              Cbo--D-Nval--OpNP                                                                         82.7  H 5.80 5.78 1.00:1.00:0.98                                                    N 16.74                                                                              16.49                                                                  Br                                                                              11.62                                                                              11.76                         32 Benzenesulfonyl-D-Leu--Gly--                                                                      7f (5 mMole)                                                                              Example 7g                                                                          C 45.85                                                                              45.55                                                                              Gly:Leu:Arg                 Arg--pNA.HBr        Benzenesulfo-                                                                             92    H 5.47 5.44 1.00:1.01:0.99              C.sub.26 H.sub.37 N.sub.8 O.sub.7 SBr                                                             chloride          N 16.58                                                                              16.34                                                (5 mMole)         S 4.53 4.68                                                                   Br                                                                              11.30                                                                              11.65                         33 Methanesulfonyl-D-Leu--Gly--                                                                      7f (5 mMole)                                                                              Example 7g                                                                          C 40.90                                                                              40.45                                                                              Gly:Leu:Arg                 Arg--pNA.HBr        Methanesulfo-                                                                             90.5  H 5.71 5.66 1.00:1.02:0.99              C.sub.21 H.sub.35 N.sub.8 O.sub.7 SBr                                                             chloride          N 18.28                                                                              17.97                                                (5 mMole)         S 5.01 5.14                                                                   Br                                                                              12.55                                                                              12.82                         34 α-Naphthalenesulfonyl-D-Leu--                                                               7f (5 mMole)                                                                              Example 7g                                                                          C 49.38                                                                              48.98                                                                              Gly:Leu:Arg                 Gly--Arg--pNA.HBr   α-Naphthalene-                                                                      88    H 5.40 5.34 1.00:1.01:0.98              C.sub.30 H.sub.39 N.sub.8 O.sub.7 SBr                                                             sulfochloride     N 15.55                                                                              15.23                                                (5 mMole)         S 4.25 4.36                                                                   Br                                                                              10.62                                                                              10.86                         35 n-Butanesulfonyl-D-Leu--Gly--                                                                     7f (5 mMole)                                                                              Example 7g                                                                          C 43.55                                                                              43.31                                                                              Gly:Leu:Arg                 Arg--pNA.HBr        n-Butanesulfo-                                                                            88.3  H 6.28 6.21 1.00:0.99:0.99              C.sub.24 H.sub.41 N.sub.8 O.sub.7 SBr                                                             chloride          N 17.08                                                                              16.84                                                (5 mMole)         S 4.73 4.82                                                                   Br                                                                              11.84                                                                              12.00                         36 Ac--D-Nleu--Gly--Arg--pNA.HBr                                                                     36f (5 mMole)                                                                             Example 7g                                                                          C 45.18                                                                              44.98                                                                              Gly:Nleu:Arg                C.sub.22 H.sub.35 N.sub.8 O.sub.6 Br                                                              Acetic acid 78.5  H 6.08 6.01 1.00:1.01:1.00                                  anhydride         N 19.38                                                                              19.07                                                (6 mMole)         Br                                                                              13.35                                                                              13.60                         37 n-Butyryl-D-Nleu--Gly--Arg--                                                                      36f (5 mMole)                                                                             Example 7g                                                                          C 47.03                                                                              46.83                                                                              Gly:Nleu:Arg                pNA.HBr             n-Butyryl-  81.2  H 6.45 6.39 1.00:0.97:0.99              C.sub.24 H.sub.39 N.sub.8 O.sub.6 Br                                                              chloride          N 18.41                                                                              18.21                                                (5 mMole)         Br                                                                              15.38                                                                              15.60                         38 n-Octanoyl-D-Nleu--Gly--Arg--                                                                     36f (5 mMole)                                                                             Example 7g                                                                          C 49.88                                                                              50.07                                                                              Gly:Leu:Arg                 pNA.HBr             n-Octanoyl- 78.1  H 7.11 7.01 1.00:1.01:0.98              C.sub.28 H.sub.47 N.sub.8 O.sub.6 Br                                                              chloride          N 16.79                                                                              16.68                                                (5 mMole)         Br                                                                              11.65                                                                              11.90                         39 2-Phenylacetyl-D-Leu--Sar--                                                                       39f (5 mMole)                                                                             Example 7g                                                                          C 51.62                                                                              51.40                                                                              Sar:Leu:Arg                 Arg--pNA.HBr        2--Phenylacetic                                                                           80.4  H 6.12 6.10 1.00:1.02:1.00              C.sub.29 H.sub.41 N.sub.8 O.sub.6 Br                                                              acid-OpNP         N 16.80                                                                              16.54                                                (5.5 mMole)       Br                                                                              11.53                                                                              11.79                         40 4-Phenylbutyryl-D-Leu--Sar--                                                                      39f (5 mMole)                                                                             Example 7g                                                                          C 52.99                                                                              52.76                                                                              Sar:Leu:Arg                 Arg--pNA.HBr        4-Phenylbutyric                                                                           79.2  H 6.47 6.43 1.00:0.99:0.98              C.sub.31 H.sub.45 N.sub.8 O.sub.6 Br                                                              acid-OpNP         N 16.05                                                                              15.88                                                (5.5 mMole)       Br                                                                              11.18                                                                              11.32                         41 Cyclohexylcarbonyl-D-Leu--                                                                        39f (5 mMole)                                                                             Example 7g                                                                          C 50.51                                                                              50.22                                                                              Sar:Leu:Arg                 Sar--Arg--pNA.HBr   Cyclohexanecarb-                                                                          82.3  H 6.81 6.77 1.00:1.01:1.00              C.sub.28 H.sub.45 N.sub.8 O.sub.6 Br                                                              oxylic acid-OpNP  N 16.75                                                                              16.73                                                (5.5 mMole)       Br                                                                              11.81                                                                              11.93                         42 Cbo--4-aminomethyl-cyclohexyl-                                                                    39f (5 mMole)                                                                             Example 7g                                                                          C 53.68                                                                              53.36                                                                              Sar:Leu:Arg                 carbonyl-D-Leu--Sar--Arg--pNA.                                                                    Cbo--4-aminome-                                                                           84.1  H 5.56 6.54 1.00:0.99:0.98              C.sub.37 H.sub.54 N.sub.9 O.sub.8 Br HBr                                                          thylhexanecarbo-  N 15.30                                                                              15.14                                                xylic acid-OpNP   Br                                                                              9.31 9.59                                                 (5 mMole)                                              43 4-Aminomethyl-cyclohexyl-car-                                                                     42 (3 mMole)                                                                              Example 1d                                                                          C 44.51                                                                              44.68                                                                              Sar:Leu:Arg                 bonyl-D-Leu--Sar--Arg--pNA.2HBr                                                                   2H HBr/AcOH 96.2  H 6.39 6.34 1.00:0.99:0.98              C.sub.29 H.sub.49 N.sub.9 O.sub.6 Br.sub.2                                                                          N 16.38                                                                              16.17                                                                  Br                                                                              20.18                                                                              20.50                         44 Cbo--Gly--D-Nleu--Sar--Arg--pNA.                                                                  44f (5 mMole)                                                                             Example 1c                                                                          C 49.88                                                                              49.60                                                                              Sar:Nleu:Arg                HBr                 Cbo--Gly--OpNP                                                                            77.8  H 5.96 5.91 1.00:0.98:0.99              C.sub.31 H.sub.44 N.sub.9 O.sub.8 Br                                                              (5.5 mMole)       N 16.93                                                                              16.79                                                                  Br                                                                              10.44                                                                              10.64                         45 H--Gly--D-Nleu--Sar--Arg--pNA.                                                                    44 (3 mMole)                                                                              Example 1d                                                                          C 39.88                                                                              39.61                                                                              Sar:Nleu:Arg                2HBr                2N HBr/AcOH 93.6  H 5.69 5.64 1.00:1.00:0.98              C.sub.23 H.sub.39 N.sub.9 O.sub.6 Br.sub.2                                                                          N 18.18                                                                              18.08                                                                  Br                                                                              22.52                                                                              22.91                         46 Cbo--ω-Aminobutyryl-D-Nleu--                                                                44f (5 mMole)                                                                             Example 7g                                                                          C 50.49                                                                              50.90                                                                              Sar:Nleu:Arg                Sar--Arg--pNA.HBr   Cbo--4-Amino-                                                                             77.2  H 6.26 6.21 1.00:0.99:0.98              C.sub.33 H.sub.48 N.sub.9 O.sub.8 Br                                                              butyryl-OpNP      N 16.39                                                                              16.19                                                (5.5 mMole)       Br                                                                              10.11                                                                              10.26                         47 ω-Aminobutyryl-D-Nleu--Sar--                                                                46 (3 mMole)                                                                              Example 1d                                                                          C 41.65                                                                              41.39                                                                              Sar:Nleu:Arg                Arg--pNA.2HBr       2N HBr/AcOH 90.8  H 6.03 5.97 1.00:1.01:0.98              C.sub.25 H.sub.43 N.sub.9 O.sub.6 Br.sub.2                                                                          N 17.39                                                                              17.38                                                                  Br                                                                              21.75                                                                              22.03                         48 Cbo--ω-Aminooctanoyl-D-Nleu--                                                               44f (5 mMole)                                                                             Example 7g                                                                          C 53.09                                                                              53.23                                                                              Sar:Nleu:Arg                Sar--Arg--pNA.HBr   Cbo--8-Aminooc-                                                                           76.2  H 6.80 6.76 1.00:0.98:0.99              C.sub.37 H.sub.56 N.sub.9 O.sub.8 Br                                                              tanoyl-OpNP       N 14.88                                                                              15.10                                                (5.5 mMole)       Br                                                                              9.45 9.57                          49 ω-Aminooctanoyl--Sar--                                                                      48 (3 mMole)                                                                              Example 1d                                                                          C 44.95                                                                              44.56                                                                              Sar:Nleu:Arg                Arg--pNA.2HBr       2N HBr/AcOH 92.8  H 6.59 6.58 1.00:0.98:1.00              C.sub.29 H.sub.51 N.sub.9 O.sub.6 Br  N 16.31                                                                              16.13                                                                  Br                                                                              20.12                                                                              20.45                         50 Bz--D-Nval--Gly--Arg--pNA.HBr                                                                     50f (5 mMole)                                                                             Example 7g                                                                          C 49.25                                                                              49.14                                                                              Gly:Nval:Arg                C.sub.26 H.sub.35 N.sub.8 O.sub.6 Br                                                              Benzoic acid an-                                                                          77.5  H 5.59 5.55 1.00:0.99:0.98                                  hydride (Bz.sub.2 O)                                                                            N 17.90                                                                              17.63                                                (7.5 mMole)       Br                                                                              12.33                                                                              12.57                         51 4-Methylbenzoyl-D-Nval--Gly--                                                                     50f (5 mMole)                                                                             Example 7g                                                                          C 50.18                                                                              49.93                                                                              Gly:Nval:Arg                Arg--pNA.HBr        4-Methylbenzoic                                                                           76.8  H 5.78 5.74 1.00:1.01:0.98              C.sub.27 H.sub.37 N.sub.8 O.sub.6 Br                                                              acid anhydride    N 17.35                                                                              17.25                                                (7.5 mMole)       Br                                                                              12.18                                                                              12.30                         52 2-Chlorobenzoyl-D-Nval--Gly--                                                                     50f (5 mMole)                                                                             Example 7g                                                                          C 50.33                                                                              49.92                                                                              Gly:Nval:Arg                Arg--pNA.HCl        2-Chlorobenzoic                                                                           76.4  H 5.46 5.48 1.00:0.99:0.99              C.sub.26 H.sub.34 N.sub.8 O.sub.6 Cl.sub.2                                                        acid anhydride    N 18.09                                                                              17.91                                                (7.5 mMole)       Cl                                                                              11.18                                                                              11.34                         53 2-(4-Cbo--amino)-phenylace-                                                                       50f (5 mMole)                                                                             Example 7g                                                                          C 52.48                                                                              52.63                                                                              Gly:Nval:Arg                tyl-D-Nval--Gly--Arg--pNA.HBr                                                                     2-(4-Cbo--amino)-                                                                         77.9  H 5.52 5.55 1.00:0.98:0.98              C.sub.35 H.sub.44 N.sub.9 O.sub.8 Br                                                              Phenylacetyl-OpNP N 15.94                                                                              15.78                                                (5.5 mMole)       Br                                                                              9.78 10.00                         54 2-(4-Amino)-phenylacetyl-D-                                                                       53 (3 mMole)                                                                              Example 1d                                                                          C 43.17                                                                              43.50                                                                              Gly:Nval:Arg                Nval--Gly--Arg--pNA.2HBr                                                                          2N HBr/AcOH 91.8  H 5.24 5.27 1.00:0.99:1.00              C.sub.35 H.sub.44 N.sub.9 O.sub.8 Br.sub.2                                                                          N 17.09                                                                              16.91                                                                  Br                                                                              21.18                                                                              21.44                         55 CH.sub.3 O--CO--D-Nval--Sar--Arg--pNA.                                                            55f (5 mMole)                                                                             Example 7g                                                                          C 43.59                                                                              43.79                                                                              Sar:Nval:Arg                HBr                 Methyl chloro-                                                                            83.6  H 5.92 5.85 1.00:0.98:0.99              C.sub.22 H.sub.35 N.sub.8 O.sub.7 Br                                                              formate           N 18.76                                                                              18.57                                                (5.5 mMole)       Br                                                                              12.95                                                                              13.24                         56 CH.sub.3 --(CH.sub.2).sub.7 --O--CO--D-Nval--Sar--                                                55f (5 mMole)                                                                             Example 7g                                                                          C 49.51                                                                              49.64                                                                              Sar:Nval:Arg                Arg--pNA.HBr        Octyl       79.8  H 7.08 7.04 1.00:1.00:0.98              C.sub.29 H.sub.49 N.sub.8 O.sub.7 Br                                                              chloroformate     N 16.22                                                                              15.97                                                (5.5 mMole)       Br                                                                              11.19                                                                              11.39                         57 BOC--D-Leu--Gly--Arg--pNA.HBr                                                                     1d (5 mMole)                                                                              Example 1e                                                                          C 46.88                                                                              46.51                                                                              Gly:Leu:Arg                 C.sub.25 H.sub.41 N.sub.8 O.sub.7 Br                                                              BOC--D-Leu--OpNP                                                                          84.2  H 6.41 6.40 1.00:1.00:0.99                                  (5.5 mMole)       N 17.71                                                                              17.36                                                                  Br                                                                              12.20                                                                              12.38                         58 4-MeO--Cbo--D-Leu--Gly--Arg--pNA.                                                                 1d (5 mMole)                                                                              Example 1e                                                                          C 48.81                                                                              49.09                                                                              Gly:Leu:Arg                 HBr                 4-MeO--Cbo--D-                                                                            80.5  H 5.90 5.82 1.00:1.01:0.98              C.sub.29 H.sub.41 N.sub.8 O.sub.8 Br                                                              Leu--OpNP         N 16.00                                                                              15.79                                                (5.5 mMole)       Br                                                                              11.05                                                                              11.26                         59 CH.sub.3 O--CO--D-CHA--Gly--Arg--pNA.                                                             59f (5 mMole)                                                                             Examples                                                                            C 52.60                                                                              52.08                                                                              Gly:CHA:Arg                 AcOH                CH.sub.3 O--CO--Cl                                                                        7 & 6 H 6.88 6.80 1.00:0.97:0.99              C.sub.27 H.sub.42 N.sub.8 O.sub.9                                                                 (5.5 mMole) 85.6  N 18.30                                                                              18.00                         60 C.sub.2 H.sub.5 O--CO--D-CHA--Gly--Arg--                                                          59f (5 mMole)                                                                             Examples                                                                            C 52.48                                                                              52.82                                                                              Gly:CHA:Arg                 pNA.AcOH            C.sub.2 H.sub.5 O--CO--Cl                                                                 7 & 6 H 7.03 6.97 1.00:0.98:0.98              C.sub.28 H.sub.44 N.sub.8 O.sub.9                                                                 (5.5 mMole) 84.3  N 17.75                                                                              17.60                         61 CH.sub.3 SO.sub.2 --D-CHA--Gly--Arg--                                                             59f (5 mMole)                                                                             Examples                                                                            C 48.22                                                                              48.59                                                                              Gly:CHA:Arg                 pNA.AcOH            CH.sub.3 SO.sub.2 --Cl                                                                    7 & 6 H 6.66 6.59 1.00:0.97:0.97              C.sub.26 H.sub.42 N.sub.8 O.sub.9 S                                                               (5.5 mMole) 86.8  N 17.58                                                                              17.44                                                                  S 4.80 4.99                          62 Cbo--D-Leu--Gly--Arg--2-NA.                                                                       62d (5 mMole)                                                                             Example 1e                                                                          C 55.78                                                                              56.14                                                                              Gly:Leu:Arg                 HBr                 Cbo--D-Leu--OpNP                                                                          79.5  H 6.24 6.18 1.00:1.01:0.99              C.sub.32 H.sub.42 N.sub.7 O.sub.5 Br                                                              (5.5 mMole)       N 14.48                                                                              14.32                                                                  Br                                                                              11.45                                                                              11.67                         63 CH.sub.3 O--CO--D-Leu--Gly--Arg--                                                                 62f (5 mMole)                                                                             Examples                                                                            C 57.02                                                                              57.23                                                                              Gly:Leu:Arg                 2-NA.AcOH           CH.sub.3 O--CO--Cl                                                                        7 & 6 H 7.10 7.03 1.00:1.00:0.98              C.sub.28 H.sub.41 N.sub.7 O.sub.7                                                                 (5.5 mMole) 75.9  N 16.83                                                                              16.68                         64 CH.sub.3 SO.sub.2 --D-Leu--Gly--Arg--                                                             62f (5 mMole)                                                                             Examples                                                                            C 53.08                                                                              53.36                                                                              Gly:Leu:Arg                 2-NA.AcOH           CH.sub.3 SO.sub.2 --Cl                                                                    7 & 6 H 6.84 6.80 1.00:1.01:0.98              C.sub.27 H.sub.41 N.sub.7 O.sub.7 S                                                               (5 mMole)   82.1  N 16.25                                                                              16.13                                                                  S 5.15 5.28                          65 Cbo--D-Leu--Gly--Arg--4-                                                                          65d (5 mMole)                                                                             Example 1e                                                                          C 55.16                                                                              55.46                                                                              Gly:Leu:Arg                 MeO--2-NA.HBr       Cbo--D-Leu--OpNP                                                                          84.0  H 6.25 6.21 1.00:1.01:0.99              C.sub.33 H.sub.44 N.sub.7 O.sub.6 Br                                                              (5.5 mMole)       N 13.96                                                                              13.72                                                                  Br                                                                              11.00                                                                              11.18                         66 CH.sub.3 O--CO--D-Leu--Gly--Arg--                                                                 65f (5 mMole)                                                                             Examples                                                                            C 56.18                                                                              56.39                                                                              Gly:Leu:Arg                 4-MeO--2-NA.AcOH    CH.sub.3 O--CO--Cl                                                                        7 & 6 H 7.08 7.02 1.00:0.99:0.99              C.sub.29 H.sub.43 N.sub.7 O.sub.8                                                                 (5.5 mMole) 82.4  N 16.09                                                                              15.87                         67 CH.sub.3 SO.sub.2 --D-Leu--Gly--Arg--                                                             65f (5 mMole)                                                                             Examples                                                                            C 52.58                                                                              52.73                                                                              Gly:Leu:Arg                 4-MeO--2-NA.AcOH    CH.sub.3 SO.sub.2 Cl                                                                      7 & 6 H 6.85 6.80 1.00:1.01:0.98              C.sub.28 H.sub.43 N.sub.7 O.sub.8 S                                                               (5.5 mMole) 77.4  N 15.58                                                                              15.37                                                                  S 4.92 5.03                          68 CH.sub.3 O--CO--D-Leu--Gly--Arg--                                                                 68f (5 mMole)                                                                             Examples                                                                            C 51.23                                                                              51.45                                                                              Gly:Leu:Arg                 DPA.AcOH            CH.sub.3 O--CO--Cl                                                                        7 & 6 H 6.65 6.63 1.00:0.99:0.99              C.sub.28 H.sub.43 N.sub.7 O.sub.11                                                                (5.5 mMole) 79.3  N 15.18                                                                              15.00                         69 CH.sub.3 SO.sub.2 --D-Leu--Gly--Arg--                                                             68f (5 mMole)                                                                             Examples                                                                            C 47.88                                                                              48.13                                                                              Gly:Leu:Arg                 DPA.AcOH            CH.sub.3 SO.sub.2 --Cl                                                                    7 & 6 H 6.50 6.43 1.00:1.00:0.98              C.sub.27 H.sub.43 N.sub.7 O.sub.11 S                                                              (5.5 mMole) 81.2  N 14.75                                                                              14.55                                                                  S 4.68 4.76                          70 CH.sub.3 O--CO--D-Leu--Gly--Arg--                                                                 70f (5 mMole)                                                                             Examples                                                                            C 54.48                                                                              54.27                                                                              Gly:Leu:Arg                 MCA.AcOH            CH.sub.3 O--CO--Cl                                                                        7 & 6 H 6.72 6.67 1.00:1.01:1.00              C.sub.28 H.sub.41 N.sub.7 O.sub.9                                                                 (5.5 mMole) 83.2  N 16.03                                                                              15.82                         71 CH.sub.3 SO.sub.2 --D-Leu--Gly--Arg--                                                             70f (5 mMole)                                                                             Examples                                                                            C 50.93                                                                              50.69                                                                              Gly:Leu:Arg                 MCA.AcOH            CH.sub.3 SO.sub.2 --Cl                                                                    7 & 6 H 6.51 6.46 1.00:0.99:0.98              C.sub.27 H.sub.41 N.sub.7 O.sub.9 S                                                               (5.5 mMole) 78.4  N 15.53                                                                              15.33                                                                  S 4.92 5.01                          72 Cbo--D-Leu--Gly--Arg--5-                                                                          72d (5 mMole)                                                                             Example                                                                             C 52.88                                                                              52.68                                                                              Gly:Leu:Arg                 NO.sub.2 --1-NA.HBr Cbo--D-Leu--OpNP                                                                          1e    H 5.69 5.66 1.00:1.01:0.99              C.sub.32 H.sub.41 N.sub.8 O.sub.7 Br                                                              (5.5 mMole) 75.8  N 15.53                                                                              15.36                                                                  Br                                                                              10.82                                                                              10.95                         73 CH.sub.3 SO.sub.2 --D-Leu--Gly--Arg--                                                             72f (5 mMole)                                                                             Examples                                                                            C 50.08                                                                              49.68                                                                              Gly:Leu:Arg                 5-NO.sub.2 --1-NA.AcOH                                                                            CH.sub.3 SO.sub.2 --Cl                                                                    7 & 6 H 6.24 6.18 1.00:1.00:0.97              C.sub.27 H.sub.40 N.sub.8 O.sub.9 S                                                               (5.5 mMole) 80.8  N 17.29                                                                              17.17                                                                  S 4.82 4.91                          74 Cbo--D-Leu--Gly--Arg--5-                                                                          74d (5 mMole)                                                                             Example 1e                                                                          C 54.28                                                                              54.31                                                                              Gly:Leu:Arg                 ChA.HBr             Cbo--D-Leu--OpNP                                                                          78.5  H 6.07 6.03 1.00:0.98:0.98              C.sub.31 H.sub.41 N.sub.8 O.sub.5 Br                                                              (5.5 mMole)       N 16.48                                                                              16.34                                                                  Br                                                                              11.54                                                                              11.65                         75 CH.sub.3 SO.sub.2 --D-Leu--Gly--Arg--                                                             74f (5 mMole)                                                                             Examples                                                                            C 51.68                                                                              51.30                                                                              Gly:Leu:Arg                 5-ChA.AcOH          CH.sub.3 SO.sub.2 --Cl                                                                    7 & 6 H 6.67 6.62 1.00:0.99:0.98              C.sub.26 H.sub.40 N.sub.7 O.sub.4 S                                                               (5.5 mMole) 76.5  N 18.52                                                                              18.41                                                                  S 5.18 5.27                          76 Cbo--D-Leu--Gly--Arg--8-                                                                          76d (5 mMole)                                                                             Example 1e                                                                          C 51.11                                                                              50.96                                                                              Gly:Leu:Arg                 NO.sub.2 -- 5-ChA.HBr                                                                             Cbo--D-Leu--OpNP                                                                          79.0  H 5.54 5.52 1.00:0.98:0.97              C.sub.31 H.sub.40 N.sub.9 O.sub.7 Br                                                              (5.5 mMole)       N 17.48                                                                              17.25                                                                  Br                                                                              10.76                                                                              10.94                         77 CH.sub.3 SO.sub.2 --D-Leu--Gly--Arg--8-                                                           76f (5 mMole)                                                                             Examples                                                                            C 48.03                                                                              47.77                                                                              Gly:Leu:Arg                 NO.sub.2 --5-ChA.AcOH                                                                             CH.sub.3 SO.sub.2 --Cl                                                                    7 & 6 H 6.07 6.01 1.00:0.98:0.97              C.sub.26 H.sub.39 N.sub.9 O.sub.9 S                                                               (5.5 mMole) 75.7  N 19.44                                                                              19.28                                                                  S 4.83 4.91                          78 BOC--D-Nval--But--Arg--                                                                           78d (5 mMole)                                                                             Example 1e                                                                          C 47.45                                                                              47.34                                                                              Nval:But:Arg                pNA.HBr             BOC--D-Nval--OpNP                                                                         74.6  H 6.61 6.57 1.00:0.98:0.98              C.sub.26 H.sub.43 N.sub.8 O.sub.7 Br                                                              (5.5 mMole)       N 17.24                                                                              16.99                                                                  Br                                                                              11.88                                                                              12.11                         79 BOC--D-Nval--N(n-Bu)Gly--                                                                         79d (5 mMole)                                                                             Example 1e                                                                          C 49.08                                                                              48.91                                                                              Nval:Arg                    Arg--pNA.HBr        BOC--D-Nval--OpNP                                                                         66.3  H 6.95 6.89 1.00:0.98                   C.sub.28 H.sub.47 N.sub.8 O.sub.7 Br                                                              (5.5 mMole)       N 16.53                                                                              16.30                                                                  Br                                                                              11.41                                                                              11.62                         80 β-Naphthyl-SO.sub.2 D-Leu--                                                                  7f (5 mMole)                                                                              Example 7                                                                           C 49.25                                                                              48.98                                                                              Gly:Leu:Arg                 Gly--Arg--pNA.HBr   β-NA--SO.sub.2 --Cl                                                                  80.2  H 5.41 5.34 1.00:0.98:0.97              C.sub.30 H.sub.39 N.sub.8 O.sub.7 SBr                                                             (5.5 mMole)       N 15.43                                                                              15.23                                                                  Br                                                                              10.70                                                                              10.86                                                                  S 4.15 4.36                          81 4-NH.sub.2 Bz--D-Nleu--Gly--                                                                      36f (5 mMole)                                                                             Examples                                                                            C 43.68                                                                              43.50                                                                              Gly:Nleu:Arg                Arg--pNA.2HBr       (HCl)p-NH.sub.2 --                                                                        7 & 6 H 5.32 5.27 1.00:1.02:0.98              C.sub.27 H.sub.39 N.sub.9 O.sub.6 Br.sub.2                                                        Bz--Cl      65.0  N 17.13                                                                              16.91                                                (5.5 mMole)       Br                                                                              21.05                                                                              21.44                         82 4-Me--Cbo--D-Leu--Gly--Arg--                                                                      1d (5 mMole)                                                                              Examples                                                                            C 55.68                                                                              55.35                                                                              Gly:Leu:Arg                 pNA.AcOH            4-Me--Cbo--D-Leu--                                                                        1e & 6                                                                              H 6.64 6.59 1.00:1.01:0.99              C.sub.31 H.sub.44 N.sub.8 O.sub.9                                                                 OpNP        75.0  N 16.85                                                                              16.66                                                (5.5 mMole)                                            83 4-Cl--Cbo--D-Leu--Gly--Arg--                                                                      1d (5 mMole)                                                                              Examples                                                                            C 52.18                                                                              51.98                                                                              Gly:Leu:Arg                 pNA.AcOH            4-Cl--Cbo--D-                                                                             1e & 6                                                                              H 5.99 5.96 1.00:1.01:0.98              C.sub.30 H.sub.41 N.sub.8 O.sub.9 Cl                                                              Leu--OpNP   69.3  N 16.25                                                                              16.17                                                (5.5 mMole)       Cl                                                                              5.03 5.12                          84 BOC-- D-(α)-AOA--Gly--Arg--                                                                 1d (5 mMole)                                                                              Examples                                                                            C 53.45                                                                              53.36                                                                              Gly:Arg                     pNA.AcOH            BOC--D-(α)-AOA--                                                                    1e & 6                                                                              H 7.51 7.41 1.00:0.99                   C.sub.29 H.sub.48 N.sub.8 O.sub.9                                                                 OpNP        65.4  N 17.38                                                                              17.17                                                (5.5 mMole)                                            85 Cbo--D-Tyr(OBzl)--Gly--                                                                           1d (5 mMole)                                                                              Example                                                                             C 55.26                                                                              55.68                                                                              Gly:Tyr:Arg                 Arg--pNA.HBr        Cbo--D-Tyr(OBzl)--                                                                        1e    H 5.33 5.29 1.00:0.97:0.99              C.sub.38 H.sub.43 N.sub.8 O.sub.8 Br                                                              OpNP        75.5  N 13.85                                                                              13.67                                                (5.5 mMole)       Br                                                                              9.58 9.75                          86 CH.sub.3 O--CO--D-Tyr--Gly--Arg--                                                                 85f (5 mMole)                                                                             Examples                                                                            C 51.09                                                                              51.26                                                                              Gly:Tyr:Arg                 pNA.AcOH            CH.sub.3 O--CO--Cl                                                                        7 & 6 H 5.77 5.74 1.00:0.98:1.01              C.sub.27 H.sub.36 N.sub.8 O.sub.10                                                                (5.5 mMole) 58.4  N 17.90                                                                              17.71                         87 Cbo--D-Ser(OtBu)--Gly--                                                                           1d (5 mMole)                                                                              Examples                                                                            C 53.78                                                                              54.06                                                                              Gly:Ser:Arg                 Arg--pNA.AcOH       Cbo--D-Ser(OtBu)                                                                          1e & 6                                                                              H 6.48 6.44 1.00:0.96:0.99              C.sub.31 H.sub.44 N.sub.8 O.sub.10                                                                --OpNP      73.8  N 16.45                                                                              16.27                                                (5.5 mMole)                                            88 Cbo--D-Ser--Gly--Arg--pNA.                                                                        87 (5 mMole)                                                                              Examples                                                                            C 51.00                                                                              51.26                                                                              Gly:Ser:Arg                 AcOH                CF.sub.3 COOH                                                                             1d & 6                                                                              H 5.79 5.74 1.00:0.97:1.00              C.sub.27 H.sub.36 N.sub.8 O.sub.10                                                                (10 mMole)  85.3  N 18.02                                                                              17.71                         89 Cbo--D-Ser(OMe)--Gly--Arg--                                                                       1d (5 mMole)                                                                              Examples                                                                            C 51.75                                                                              52.01                                                                              Gly:Arg                     pNA.AcOH            Cbo--D-Ser(OMe)--                                                                         1e & 6                                                                              H 6.00 5.92 1.00:0.98                   C.sub.28 H.sub.38 N.sub.8 O.sub.10                                                                OpNP        74.6  N 17.47                                                                              17.33                                                (5.5 mMole)                                            90 Cbo--D-Ser(OBzl)--Gly--                                                                           1d (5 mMole)                                                                              Examples                                                                            C 55.95                                                                              56.50                                                                              Gly:Arg                     Arg--pNA.AcOH       Cbo--D-Ser(OBzl)                                                                          1e & 6                                                                              H 5.91 5.86 1.00:0.99                   C.sub.34 H.sub.42 N.sub.8 O.sub.10                                                                --OpNP      80.4  N 15.68                                                                              15.50                                                (5.5 mMole)                                            91 Cbo--D-Thr(OtBu)--Gly--                                                                           1d (5 mMole)                                                                              Examples                                                                            C 54.19                                                                              54.69                                                                              Gly:Thr:Arg                 Arg--pNA.AcOH       Cbo--D-     1e & 6                                                                              H 6.66 6.60 1.00:0.96:0.98              C.sub.32 H.sub.46 N.sub.8 O10                                                                     Thr(OtBu)--OpNP                                                                           78.4  N 16.17                                                                              15.95                                                (5.5 mMole)                                            92 Cbo--D-Thr--Gly--Arg--pNA.                                                                        91 (5 mMole)                                                                              Examples                                                                            C 51.88                                                                              52.01                                                                              Gly:Thr:Arg                 AcOH                CF.sub.3 COOH                                                                             1d & 6                                                                              H 5.95 5.92 1.00:0.97:0.98              C.sub.28 H.sub.38 N.sub.8 O.sub.10                                                                (10 mMole)  82.0  N 17.45                                                                              17.33                         93 Cbo--D-Thr(OMe)--Gly--Arg--                                                                       1d (5 mMole)                                                                              Examples                                                                            C 52.15                                                                              52.72                                                                              Gly:Arg                     pNA.AcOH            Cbo--D-Thr(OMe)--                                                                         1e & 6                                                                              H 6.13 6.10 1.00:0.98                   C.sub.29 H.sub.40 N.sub.8 O.sub.10                                                                OpNP        75.5  N 17.20                                                                              16.96                                                (5.5 mMole)                                            94 Cbo--D-Thr(OBzl)--Gly--                                                                           1d (5 mMole)                                                                              Examples                                                                            C 56.50                                                                              57.06                                                                              Gly:Arg                     Arg--pNA.AcOH       Cbo--D-Thr(OBzl)--                                                                        1e & 6                                                                              H 6.07 6.02 1.00:1.00                   C.sub.35 H.sub.44 N.sub.8 O.sub.10                                                                OpNP        81.2  N 15.45                                                                              15.21                                                (5.5 mMole)                                            95 Cbo--D-Asp--Gly--Arg--                                                                            20 (5 mMole)                                                                              Examples                                                                            C 51.66                                                                              52.00                                                                              Gly:Asp:Arg                 pNA                 CF.sub.3 COOH                                                                             1d & 6                                                                              H 5.43 5.37 1.00:0.98:1.01              C.sub.26 H.sub.32 N.sub.8 O.sub.9                                                                 (15 mMole)  85.0  N 18.95                                                                              18.66                         96 Cbo--D-Glu--Gly--Arg--                                                                            19 (5 mMole)                                                                              Examples                                                                            C 52.21                                                                              52.76                                                                              Gly:Glu:Arg                 pNA                 CF.sub.3 COOH                                                                             1d & 6                                                                              H 5.61 5.58 1.00:0.97:0.99              C.sub.27 H.sub.34 N.sub.8 O.sub.9                                                                 (15 mMole)  82.4  N 18.48                                                                              18.23                         97 Cbo--D-Glu(γ-OMe)--Gly--Arg--                                                               1d (5 mMole)                                                                              Examples                                                                            C 51.84                                                                              52.32                                                                              Gly:Glu:Arg                 pNA.AcOH            Cbo--D-Glu(γ-                                                                       1e & 6                                                                              H 5.89 5.85 1.00:1.01:0.98              C.sub.30 H.sub.40 N.sub.8 O.sub. 11                                                               OMe)--OpNP  78.6  N 16.47                                                                              16.27                                                (5.5 mMole)                                            98 Cbo--D-Asp(β-OMe)--Gly--                                                                     1d (5 mMole)                                                                              Examples                                                                            C 51.48                                                                              51.63                                                                              Gly:Asp:Arg                 Arg--pNA.AcOH       Cbo--D-Asp(β-                                                                        1e & 6                                                                              H 5.71 5.68 1.00:1.02:1.00              C.sub.29 H.sub.38 N.sub.8 O.sub.11                                                                OMe)--OpNP  78.9  N 16.93                                                                              16.61                                                (5.5 mMole)                                            99 Cbo--D-Glu(γ-OBzl)--Gly--                                                                   1d (5 mMole)                                                                              Examples                                                                            C 55.38                                                                              55.99                                                                              Gly:Glu:Arg                 Arg--pNA.AcOH       Cbo--D-Glu(γ-                                                                       1e & 6                                                                              H 5.68 5.64 1.00:0.98:0.99              C.sub.35 H.sub.42 N.sub.8 O.sub.11                                                                OBzl)--OpNP 76.7  N 15.09                                                                              14.93                                                (5.5 mMole)                                            100                                                                              Cbo--D-Asp(β-OBzl)--Gly--                                                                    1d (5 mMole)                                                                              Examples                                                                            C 55.03                                                                              55.43                                                                              Gly:Asp:Arg                 Arg--pNA.AcOH       Cbo--D-Asp(β-                                                                        1e & 6                                                                              H 5.51 5.47 1.00:0.99:0.97              C.sub.34 H.sub.40 N.sub.8 O.sub.11                                                                OBzl)--OpNP 74.8  N 15.48                                                                              15.21                                                (5.5 mMole)                                            101                                                                              Cbo--D-Aadi(δ-OtBu)--Gly--                                                                  1d (5 mMole)                                                                              Examples                                                                            C 54.13                                                                              54.83                                                                              Gly:Arg                     Arg--pNA.AcOH       Cbo--D-Aadi(δ-                                                                      1e & 6                                                                              H 6.57 6.50 1.00:0.98                   C.sub.34 H.sub.48 N.sub.8 O.sub.11                                                                OtBu)--OpNP 71.5  N 15.29                                                                              15.05                                                (5.5 mMole)                                            102                                                                              Cbo--D-Aadi--Gly--Arg-- pNA                                                                       101 (5 mMole)                                                                             Examples                                                                            C 52.97                                                                              53.50                                                                              Gly:Arg                     C.sub.28 H.sub.36 N.sub.8 O.sub.9                                                                 CF.sub.3 COOH                                                                             1d & 6                                                                              H 5.83 5.77 1.00:0.99                                       (15 mMole)  81.1  N 18.05                                                                              17.83                         103                                                                              Cbo--D-Aadi(δ-OBzl)--Gly--                                                                  1d (5 mMole)                                                                              Examples                                                                            C 56.53                                                                              57.06                                                                              Gly:Arg                     Arg--pNA.AcOH       Cbo--D-Aadi(δ-                                                                      1e & 6                                                                              H 6.00 5.95 1.00:0.99                   C.sub.37 H.sub.46 N.sub.8 O.sub.11                                                                OBzl)--OpNP 73.9  N 14.65                                                                              14.39                                                (5.5 mMole)                                            __________________________________________________________________________

                                      TABLE 2                                     __________________________________________________________________________    Dipeptide            Starting product                                                                           Method                                                                              Elementary analysis                   Ex.                                                                              intermediate      (mMole)      yield % found %                                                                            calc. %                        __________________________________________________________________________    22c                                                                              Cbo--Sar--Arg--pNA.HBr                                                                          1b (10 mMole)                                                                              Example 1c                                                                          C 47.88                                                                              47.59                             C.sub.23 H.sub.30 N.sub.7 O.sub.6 Br                                                            Cbo--Sar--OpNP                                                                             84.1  H 5.25 5.21                                                (11 mMole)         N 17.08                                                                              16.89                                                                  Br                                                                              13.50                                                                              13.77                          22d                                                                              2HBr.H--Sar--Arg--pNA                                                                           22c (7 mMole)                                                                              Example 1d                                                                          C 34.50                                                                              34.17                             C.sub.15 H.sub.25 N.sub.7 O.sub.4 Br.sub.2                                                      2N HBr/AcOH  91.2  H 4.82 4.78                                                                   N 18.91                                                                              18.60                                                                  Br                                                                              30.06                                                                              30.31                          23c                                                                              Cbo--Ala--Arg--pNA.HBr                                                                          1b (10 mMole)                                                                              Example 1c                                                                          C 47.69                                                                              47.59                             C.sub.23 H.sub.30 N.sub.7 O.sub.6 Br                                                            Cbo--Ala--OpNP                                                                             83.6  H 5.28 5.21                                                (11 mMole)         N 16.95                                                                              16.89                                                                  Br                                                                              13.46                                                                              13.77                          23d                                                                              2HBr.H--Ala--Arg--pNA                                                                           23c (7 mMole)                                                                              Example 1d                                                                          C 34.28                                                                              34.17                             C.sub.15 H.sub.25 N.sub.7 O.sub.4 Br.sub.2                                                      2N HBr/AcOH  93.6  H 4.79 4.78                                                                   N 18.90                                                                              18.60                                                                  Br                                                                              29.88                                                                              30.31                          26c                                                                              Cbo--N(Et)Gly--Arg--pNA.HBr                                                                     1b (10 mMole)                                                                              Example 1c                                                                          C 48.62                                                                              48.49                             C.sub.24 H.sub.32 N.sub.7 O.sub.6 Br                                                            Cbo--N(Et)Gly--OpNP                                                                        86.0  H 5.48 5.43                                                (11 mMole)         N 16.55                                                                              16.49                                                                  Br                                                                              13.25                                                                              13.44                          26d                                                                              2HBr.H--N(Et)Gly--Arg--pNA                                                                      26c (7 mMole)                                                                              Example 1d                                                                          C 35.70                                                                              35.51                             C.sub.16 H.sub.27 N.sub.7 O.sub.4 Br.sub.2                                                      2N HBr/AcOH  94.0  H 5.09 5.03                                                                   N 18.30                                                                              18.12                                                                  Br                                                                              29.22                                                                              29.53                          27c                                                                              Cbo--N(Pr)Gly--Arg--pNA.HBr                                                                     1b (10 mMole)                                                                              Example 1c                                                                          C 50.08                                                                              49.35                             C.sub.25 H.sub.34 N.sub.7 O.sub.6 Br                                                            (Cbo--N(Pr)Gly--OpNP                                                                       82.5  H 5.69 5.63                                                (11 mMole)         N 16.36                                                                              16.11                                                                  Br                                                                              12.88                                                                              13.13                          27d                                                                              2HBr.H--N(Pr)Gly--Arg--pNA                                                                      27c (7 mMole)                                                                              Example 1d                                                                          C 36.68                                                                              36.77                             C.sub.17 H.sub.29 N.sub.7 O.sub.4 Br.sub.2                                                      2N HBr/AcOH  88.5  H 5.31 5.26                                                                   N 17.96                                                                              17.66                                                                  Br                                                                              28.58                                                                              28.78                          __________________________________________________________________________    Amino acid or dipeptide                                                                            Starting product                                                                           Method                                                                              Elementary analysis                   Ex.                                                                              intermediate      (mMole)      yield % found %                                                                            calc. %                        __________________________________________________________________________    62c                                                                              Cbo--Gly--Arg--2-NA.HBr                                                                         4b (5 mMole) Example 4c                                                                          C 54.24                                                                              54.64                             C.sub.26 H.sub.31 N.sub.6 O.sub.4 Br                                                            Cbo--Gly--OpNP                                                                             75.6  H 5.52 5.47                                                (5.5 mMole)        N 14.95                                                                              14.71                                                                  Br                                                                              13.75                                                                              13.98                          62d                                                                              2HBr.H--Gly--Arg--2-NA                                                                          62c (5 mMole)                                                                              Example 4d                                                                          C 41.50                                                                              41.72                             C.sub.18 H.sub.26 N.sub.6 O.sub.2 Br.sub.2                                                      2N HBr/AcOH  90.6  H 5.10 5.06                                                                   N 16.62                                                                              16.22                                                                  Br                                                                              30.15                                                                              30.84                          65c                                                                              Cbo--Gly--Arg--4-MeO--2-                                                                        5b (5 mMole) Example 5c                                                                          C 53.41                                                                              53.91                             NA.HBr            Cbo--Gly--OpNP                                                                             88.4  H 5.55 5.53                              C.sub.27 H.sub.33 N.sub.6 O.sub.5 Br                                                            (5.5 mMole)        N 14.22                                                                              13.97                                                                  Br                                                                              12.95                                                                              13.28                          65d                                                                              2HBr.H--Gly--Arg--4-MeO--                                                                       65c (5 mMole)                                                                              Example 5d                                                                          C 41.32                                                                              41.62                             2-NA              2N HBr/AcOH  90.9  H 5.19 5.15                              C.sub.19 H.sub.28 N.sub.6 O.sub.3 Br.sub.2                                                                         N 15.54                                                                              15.33                                                                  Br                                                                              28.85                                                                              29.15                          72a                                                                              Cbo--Arg--5-NO.sub.2 --1-NA.HCl                                                                 Cbo--Arg--OH.HCl                                                                           Example 3a                                                                          C 55.65                                                                              55.98                             C.sub.24 H.sub.27 N.sub.6 O.sub.5 Cl                                                            (25 mMole)   38.7  H 5.31 5.28                                                5-NO.sub.2 --1-naphthyl-                                                                         N 16.54                                                                              16.32                                               amine              Cl                                                                              6.77 6.89                                                (25 mMole)                                               72b                                                                              2HBr.H.Arg--5-NO.sub.2 --1-NA                                                                   72a (5 mMole)                                                                              Example 4b                                                                          C 37.85                                                                              37.96                             C.sub.16 H.sub.22 N.sub.6 O.sub.3 Br.sub.2                                                      2N HBr/AcOH  88.5  H 4.41 4.38                                                                   N 16.78                                                                              16.60                                                                  Br                                                                              31.10                                                                              31.57                          72c                                                                              Cbo--Gly--Arg--5-NO.sub.2 --1-NA.HBr                                                            72b (3 mMole)                                                                              Example 4c                                                                          C 50.25                                                                              50.66                             C.sub.26 H.sub.30 N.sub.7 O.sub.6 Br                                                            Cbo--Gly--OpNP                                                                             78.6  H 4.99 4.91                                                (3.3 mMole)        N 16.10                                                                              15.91                                                                  Br                                                                              12.76                                                                              12.96                          72d                                                                              2HBr.H--Gly--Arg--5-NO.sub.2 --1-NA                                                             72c (2.5 mMole)                                                                            Example 1d                                                                          C 38.17                                                                              38.38                             C.sub.18 H.sub.25 N.sub.7 O.sub.4 Br.sub.2                                                      2N HBr/AcOH  91    H 4.51 4.47                                                                   N 17.63                                                                              17.41                                                                  Br                                                                              28.00                                                                              28.37                          74a                                                                              Cbo--Arg--5-ChA.HCl                                                                             Cbo--Arg--OH.HCl                                                                           Example 3a                                                                          C 58.09                                                                              58.66                             C.sub.23 H.sub.27 N.sub.6 O.sub.3 Cl                                                            (25 mMole)   31.9  H 5.84 5.78                                                5-aminoquinoline   N 18.06                                                                              17.85                                               (25 mMole)         Cl                                                                              7.44 7.53                           74b                                                                              2HBr.H--Arg--5-ChA                                                                              74a (5 mMole)                                                                              Example 1d                                                                          C 38.75                                                                              38.98                             C.sub.15 H.sub.22 N.sub.6 OBr.sub.2                                                             2N HBr/AcOH  81.8  H 4.83 4.80                                                                   N 18.45                                                                              18.18                                                                  Br                                                                              33.88                                                                              34.58                          74c                                                                              Cbo--Gly--Arg--5-ChA.HBr                                                                        74b (3 mMole)                                                                              Example 4c                                                                          C 52.17                                                                              52.45                             C.sub.25 H.sub.30 N.sub.7 O.sub.4 Br                                                            Cbo--Gly--OpNP                                                                             68.5  H 5.32 5.28                                                (3.3 mMole)        N 17.17                                                                              17.13                                                                  Br                                                                              13.63                                                                              13.96                          74d                                                                              2HBr.H--Gly--Arg--5-ChA                                                                         74c (2.5 mMole)                                                                            Example 1d                                                                          C 39.19                                                                              39.32                             C.sub.17 H.sub.25 N.sub.7 O.sub.2 Br.sub.2                                                      2N HBr/AcOH  78.3  H 4.90 4.85                                                                   N 19.11                                                                              18.88                                                                  Br                                                                              30.45                                                                              30.78                          76a                                                                              Cbo--Arg--8-NO.sub.2 --5-ChA.HCl                                                                Cbo--Arg--OH.HCl                                                                           Example 3a                                                                          C 53.14                                                                              53.54                             C.sub.23 H.sub.26 N.sub.7 O.sub.5 Cl                                                            (25 mMole)   33.0  H 5.11 5.08                                                5-NH.sub.2 --8-NO.sub.2 --                                                                       N 19.21                                                                              19.00                                               quinoline          Cl                                                                              6.80 6.87                                                (25 mMole)                                               76b                                                                              2HBr.H--Arg--8-NO.sub.2 --5-ChA                                                                 76a (5 mMole)                                                                              Example 1d                                                                          C 35.42                                                                              35.52                             C.sub. 15 H.sub.21 N.sub.7 O.sub.3 Br.sub.2                                                     2N HBr/AcOH  84.2  H 4.20 4.17                                                                   N 19.50                                                                              19.33                                                                  Br                                                                              30.99                                                                              31.51                          76c                                                                              Cbo--Gly--Arg--8-NO.sub.2 --5-                                                                  76b (3 mMole)                                                                              Example 4c                                                                          C 48.32                                                                              48.63                             ChA.HBr           Cbo--Gly--OpNP                                                                             68.5  H 4.77 4.73                              C.sub.25 H.sub.29 N.sub.8 O.sub.6 Br                                                            (3.3 mMole)        N 18.28                                                                              18.15                                                                  Br                                                                              12.74                                                                              12.94                          76d                                                                              2HBr.H--Gly--Arg--8-NO.sub.2 --                                                                 76c (2.5 mMole)                                                                            Example 1d                                                                          C 36.00                                                                              36.19                             5-ChA             2N HBr/AcOH  76.9  H 4.33 4.29                              C.sub.17 H.sub.24 N.sub.8 O.sub.4 Br.sub.2                                                                         N 20.05                                                                              19.86                                                                  Br                                                                              27.95                                                                              28.32                          78c                                                                              Cbo--But--Arg--pNA.HBr                                                                          1b (5 mMole) Example 1c                                                                          C 48.55                                                                              48.49                             C.sub.24 H.sub.32 N.sub.7 O.sub.6 Br                                                            Cbo--But--OpNP                                                                             80.4  H 5.47 5.43                                                (5.5 mMole)        N 16.75                                                                              16.49                                                                  Br                                                                              13.20                                                                              13.44                          78d                                                                              2HBr.H--But--Arg--pNA                                                                           78c (3 mMole)                                                                              Example 1d                                                                          C 35.27                                                                              35.51                             C.sub.16 H.sub.27 N.sub.7 O.sub.4 Br.sub.2                                                      2N HBr/AcOH  84.8  H 5.03 5.03                                                                   N 18.38                                                                              18.12                                                                  Br                                                                              29.25                                                                              29.53                          79c                                                                              Cbo--N(n-Bu)Gly--Arg--pNA.HBr                                                                   1b (5 mMole) Example 1c                                                                          C 49.75                                                                              50.16                             C.sub.26 H.sub.36 N.sub.7 O.sub.6 Br                                                            Cbo--N(n-Bu)Gly--                                                                          68.2  H 5.85 5.83                                                OpNP               N 15.90                                                                              15.75                                               (5.5 mMole)        Br                                                                              12.63                                                                              12.84                          79d                                                                              2HBr.H--N(n-Bu)Gly--Arg--pNA                                                                    79c (2.5 mMole)                                                                            Example 1d                                                                          C 37.88                                                                              37.97                             C.sub.18 H.sub.31 N.sub.7 O.sub.4 Br.sub.2                                                      2N HBr/AcOH  80.7  H 5.54 5.49                                                                   N 17.50                                                                              17.22                                                                  Br                                                                              27.70                                                                              28.07                          __________________________________________________________________________

                                      TABLE 3                                     __________________________________________________________________________    Tripeptide             Starting products                                                                      Method                                                                              Elementary analysis                     Ex.                                                                              intermediates       (mMole)  yield % found %                                                                            calc. %                          __________________________________________________________________________     7f                                                                              2HBr.H--D-Leu--Gly--Arg--pNA                                                                      1 (5 mMole)                                                                            Example 2d                                                                          C 38.09                                                                              38.35                               C.sub.20 H.sub.34 N.sub.8 O.sub.5 Br.sub.2                                                        2N HBr/AcOH                                                                            96.3  H 5.41 5.47                                                                   N 17.95                                                                              17.89                                                                  Br                                                                              25.42                                                                              25.51                            36f                                                                              2HBr.H--D-Nleu--Gly--Arg--pNA                                                                     8 (5 mMole)                                                                            Example 2d                                                                          C 38.18                                                                              38.35                               C.sub.20 H.sub.34 N.sub.8 O.sub.5 Br.sub.2                                                        2N HBr/AcOH                                                                            93.4  H 5.52 5.47                                                                   N 18.06                                                                              17.89                                                                  Br                                                                              25.20                                                                              25.51                            39f                                                                              2HBr.H--D-Leu--Sar--Arg--pNA                                                                      22 (5 mMole)                                                                           Example 2d                                                                          C 39.15                                                                              39.39                               C.sub.21 H.sub.36 N.sub.8 O.sub.5 Br.sub.2                                                        2N HBr/AcOH                                                                            92.1  H 5.70 5.67                                                                   N 17.81                                                                              17.50                                                                  Br                                                                              24.81                                                                              24.96                            44f                                                                              2HBr.H--D-Nleu--Sar--Arg--pNA                                                                     30 (5 mMole)                                                                           Example 2d                                                                          C 39.08                                                                              39.39                               C.sub.21 H.sub.36 N.sub.8 O.sub.5 Br.sub.2                                                        2N HBr/AcOH                                                                            96.2  H 5.63 5.67                                                                   N 17.75                                                                              17.50                                                                  Br                                                                              24.70                                                                              24.96                            50f                                                                              2HBr.H--D-Nval--Gly--Arg--pNA                                                                     9 (5 mMole)                                                                            Example 2d                                                                          C 37.02                                                                              37.27                               C.sub.19 H.sub.32 N.sub.8 O.sub.5 Br.sub.2                                                        2N HBr/AcOH                                                                            91.8  H 5.33 5.27                                                                   N 18.55                                                                              18.30                                                                  Br                                                                              25.88                                                                              26.10                            55f                                                                              2HBr.H--D-Nval--Sar--Arg--pNA                                                                     31 (5 mMole)                                                                           Example 2d                                                                          C 38.38                                                                              38.35                               C.sub.20 H.sub.34 N.sub.8 O.sub.5 Br.sub.2                                                        2N HBr/AcOH                                                                            95.5  H 5.54 5.47                                                                   N 18.10                                                                              17.89                                                                  Br                                                                              25.25                                                                              25.51                            59f                                                                              2HBr.H--D-CHA--Gly--Arg--pNA                                                                      12 (5 mMole)                                                                           Example 1d                                                                          C 41.50                                                                              41.45                               C.sub.23 H.sub.38 N.sub.8 O.sub.5 Br.sub.2                                                        2N HBr/AcOH                                                                            82.3  H 5.77 5.75                                                                   N 17.07                                                                              16.82                                                                  Br                                                                              23.71                                                                              23.98                            62f                                                                              2HBr.H--D-Leu--Gly--Arg--2-NA                                                                     62 (5 mMole)                                                                           Example 1d                                                                          C 45.43                                                                              45.73                               C.sub.24 H.sub.36 N.sub.7 O.sub.3 Br.sub.2                                                        2N HBr/AcOH                                                                            78.5  H 5.81 5.76                                                                   N 15.81                                                                              15.55                                                                  Br                                                                              23.85                                                                              25.35                            65f                                                                              2HBr.H--D-Leu--Gly--Arg--4-                                                                       65 (5 mMole)                                                                           Example 1d                                                                          C 45.18                                                                              45.47                               MeO-- 2-NA          2N HBr/AcOH                                                                            76.9  H 5.83 5.80                                C.sub.25 H.sub.38 N.sub.7 O.sub.4 Br.sub.2                                                                       N 15.03                                                                              14.85                                                                  Br                                                                              23.85                                                                              24.20                            68f                                                                              2HBr.H--D-Leu--Gly--Arg--DPA                                                                      3e (5 mMole)                                                                           Example 1d                                                                          C 41.20                                                                              41.39                               C.sub.24 H.sub.38 N.sub.7 O.sub.7 Br.sub.2                                                        2N HBr/AcOH                                                                            76.9  H 5.55 5.50                                                                   N 14.23                                                                              14.08                                                                  Br                                                                              22.66                                                                              22.95                            70f                                                                              2HBr.H--D-Leu--Gly--Arg--MCA                                                                      2e (5 mMole)                                                                           Example 1d                                                                          C 43.17                                                                              43.52                               C.sub.24 H.sub.36 N.sub.7 O.sub.5 Br.sub.2                                                        2N HBr/AcOH                                                                            80.8  H 5.51 5.48                                                                   N 15.00                                                                              14.80                                                                  Br                                                                              23.81                                                                              24.13                            72f                                                                              2HBr.H--D-Leu--Gly--Arg--5-NO.sub.2 --                                                            72 (5 mMole)                                                                           Example 1d                                                                          C 42.51                                                                              42.68                               1-NA                2N HBr/AcOH                                                                            68.4  H 5.24 5.22                                C.sub.24 H.sub.35 N.sub.8 O.sub.5 Br.sub.2                                                                       N 16.83                                                                              16.59                                                                  Br                                                                              23.45                                                                              23.66                            74f                                                                              2HBr.H--D-Leu--Gly--Arg--5-ChA                                                                    74 (5 mMole)                                                                           Example 1d                                                                          C 43.68                                                                              43.75                               C.sub.23 H.sub.35 N.sub.8 O.sub.3 Br.sub.2                                                        2N HBr/AcOH                                                                            75.3  H 5.61 5.59                                                                   N 17.95                                                                              17.75                                                                  Br                                                                              25.08                                                                              25.31                            76f                                                                              2HBr.H--D-Leu--Gly--Arg--8-                                                                       76 (5 mMole)                                                                           Example 1d                                                                          C 40.75                                                                              40.84                               NO.sub.2 --5-ChA    2N HBr/AcOH                                                                            72.7  H 5.10 5.07                                C.sub.23 H.sub.34 N.sub.9 O.sub.5 Br.sub.2                                                                       N 18.95                                                                              18.64                                                                  Br                                                                              23.30                                                                              23.63                            85f                                                                              2(CF.sub.3 COOH).H--Tyr--Gly--Arg--pNA                                                            85 (5 mMole)                                                                           Examples                                                                            C 43.37                                                                              43.67                               C.sub.27 H.sub.32 N.sub.8 O.sub.10 F.sub.6                                                        H.sub.2 F.sub.2 (10 ml)                                                                1d & 6                                                                              H 4.35 4.34                                                             63.8  N 15.24                                                                              15.09                                                                  F 14.88                                                                              15.35                            __________________________________________________________________________

The susceptibility of the tripeptide derivatives of the invention tofactor Xa is illustrated numerically in the following Table 4 andcompared with the that of the known prior art substrateBz-Ile-Glu(γ-OH)-Gly-Arg-pNA.HCl.

                  TABLE 4                                                         ______________________________________                                        Activity of 1 ml of aqueous solution of bovine factor Xa                      and a factor Xa as obtained by activation of factor X in                      1 ml of normal human blood plasma by means of RVV, ex-                        pressed in nanomoles of split product H-- R.sup.5 released per                minute                                                                        substrate                                                                     concentration    bovine factor                                                                            human factor                                      2 × 10.sup.-4                                                                            Xa         Xa                                                ______________________________________                                        substrates according                                                          to examples                                                                    1               2560       1520                                               2               1180       760                                                3               1405       985                                                4               1290       895                                                5               1140       815                                                6               2550       1520                                               7               950        705                                                8               2780       1568                                               9               2010       1105                                              10               1332       825                                               11               246        100                                               12               1960       1528                                              13               1526       1800                                              14               1936       1548                                              15               1436       1468                                              16               1630       1320                                              17               1320       1285                                              18               1385       1235                                              19               1290       660                                               20               1045       595                                               21               1675       795                                               22               1555       1432                                              23               985        580                                               24               2214       1000                                              25               2280       820                                               26               1205       495                                               27               1025       435                                               28               2028       1656                                              29               1940       1840                                              30               1986       1608                                              31               1160       1016                                              32               1070       695                                               33               1560       987                                               34               1040       560                                               35               1380       890                                               36               210        105                                               37               280        115                                               38               295        135                                               39               980        620                                               40               825        570                                               41               600        420                                               42               30         20                                                43               60         30                                                44               105        75                                                45               250        112                                               46               205        108                                               47               380        206                                               48               180        130                                               49               410        218                                               50               550        420                                               51               560        425                                               52               555        415                                               53               680        510                                               54               740        505                                               55               980        1200                                              56               850        1060                                              57               2440       1360                                              58               2540       1430                                              59               2260       1260                                              60               3230       1420                                              61               2660       1770                                              62               1420       995                                               63               1540       1065                                              64               1520       985                                               65               1280       1025                                              66               1370       965                                               67               1410       1005                                              68               1490       1010                                              69               1515       1030                                              70               1235       795                                               71               1280       810                                               72               1180       830                                               73               1265       875                                               74               1005       660                                               75               1145       745                                               76               965        715                                               77               1080       735                                               78               795        440                                               79               850        360                                               80               940        635                                               81               495        385                                               82               2530       1380                                              83               2480       1365                                              84               2210       1690                                              85               690        580                                               86               890        610                                               87               330        146                                               88               360        165                                               89               420        170                                               90               510        225                                               91               915        830                                               92               610        415                                               93               780        630                                               94               1020       845                                               95               750        465                                               96               830        505                                               97               1880       1420                                              98               1230       1080                                              99               1990       1515                                              100              1280       1055                                              101              2080       1290                                              102              1030       905                                               103              2260       1460                                              Comparison substrate*                                                                          490        820                                               ______________________________________                                         *Bz--Ile--Glu(OH)--Gly--Arg--pNA.HCl (disclosed in German patent              application No. DEOS 25 52 570)                                          

The values of the factor Xa activity given in Table 4 were determinedexperimentally in the manner described hereinafter:

For determining the activity of bovine factor Xa 1.8 ml ofTRIS-imidazole buffer having a pH of 8.4 and an ionic strength of 0.3was well mixed at 37° C. with 0.025 ml of an aqueous solution of factorXa preparation "Diagen" (furnisher: Diagnostic Reagents Ltd., Thame,Great Britain) obtained by dissolving the contents of one vial in 0.5 mlof water. To the mixture there was added 0.2 ml of a 2×10⁻³ M aqueoussolution of a substrate of the invention. Thereafter, the quantity ofsplit product H-R⁵ in nanomoles released per minute was determined, andthe corresponding value for 1 ml of factor Xa solution was calculated.

For determining the activity of human factor Xa 0.01 ml of normalcitrated plasma was mixed with 0.20 ml of a solution of Russel vipervenom in TRIS-imidazole buffer having a pH of 8.4 and an ionic strengthof 0.3 and containing 15 mmoles of CaCl₂ per ml. The mixture wasincubated for 75 seconds at 37° C. in order to activate factor X presentin the plasma and convert it completely into factor Xa. To the incubatethere was added first 1.40 ml of TRIS-imidazole buffer (pH 8.4, ionicstrength 0.3) heated to 37° C. and then 0.40 ml of a 2×10⁻³ M aqueoussolution of a substrate of the invention. Thereafter, the quantity ofsplit product H-R⁵ in nanomoles released per minute was determined, andthe corresponding value for 1 ml of citrated plasma was calculated.

I claim:
 1. Tripeptide derivatives having the formula ##STR3## whereinR¹ represents an alkanoyl group which has 2 to 8 carbon atoms and whichmay carry an amino group in the ω-position, a phenylalkanoyl group whichhas 2 to 4 carbon atoms in the alkanoyl and the phenyl radical of whichmay be substituted with an amino group in the p-position, acyclohexylcarbonyl group which may be substituted with an aminomethylradical in the 4-position, a benzoyl group which may be substituted withmethyl, amino or halogen in the o- or p-position, an alkoxycarbonylgroup having 1 to 8 carbon atoms in the alkoxy group, abenzyloxycarbonyl group which may be substituted with methoxy, methyl orchlorine in the p-position, an alkanesulfonyl group having 1 to 4 carbonatoms, a phenylsulfonyl group which may be methylated in the p-positionor an α- or β-naphthylsulfonyl group,R² represents a straight-chained orbranched alkyl radical having 1 to 6 carbon atoms, a hydroxyalkylradical having 1 to 2 carbon atoms, an alkoxyalkyl radical having 1 to 2carbon atoms in the alkyl and 1 to 4 carbon atoms in the alkoxy, abenzyloxyalkyl radical having 1 to 2 carbon atoms in the alkyl, anω-carboxyalkyl or ω-alkoxycarbonylalkyl radical which has 1 to 3 carbonatoms in the alkyl and the alkoxy group of which is straight-chained orbranched and has 1 to 4 carbon atoms, or an ω-benzyloxycarbonyl-alkylradical having 1 to 3 carbon atoms in the alkyl, or a cyclohexyl-,cyclohexylmethyl-, 4-hydroxycyclohexylmethyl-, phenyl-, benzyl-,4-hydroxybenzyl- or imidazol-4-yl-methyl radical, except that when R² isisopropyl or cyclohexyl, R' cannot be benzyloxycarbonyl, R³ representshydrogen or a straight-chained or branched alkyl radical having 1 to 4carbon atoms, R⁴ represents hydrogen or a methyl or ethyl radical, andR⁵ represents an amino group which is substituted with aromatic orheterocyclic radicals and which is capable of being split offhydrolytically with formation of a coloured or fluorescent compoundH-R⁵, and salts thereof with acids.
 2. Tripeptide derivatives accordingto claim 1, characterized by the fact that R⁵ is a p-nitrophenylamino,1-carboxy-2-nitrophen-5-yl-amino, 1-sulfo-2-nitrophen-5-yl-amino,β-naphthylamino, 4-methoxy-β-naphthylamino, 5-nitro-α-naphthylamino,quinon-5-yl-amino, 8-nitro-quinon-5-yl-amino, 4-methylcoumar-7-yl-amino,or 1,3-di(methoxycarbonyl)-phen-5-yl-amino group (derived from5-amino-isophthalic acid dimethyl ester).
 3. Tripeptide derivativesaccording to claim 1, characterized by the fact that the strongly basicguanidino group of arginine is stabilized by protonation with a mineralor organic acid.
 4. Tripeptide derivatives according to claims 1 and 3,characterized by the fact that the guanidino group of arginine isstabilized by protonation with acetic acid.
 5. Tripeptide derivativesaccording to claim 1: Cbo-D-Leu-Gly-Arg-pNA.AcOH,Cbo-D-Leu-Sar-Arg-pNA.AcOH, Cbo-D-Ph'Gly-Gly-Arg-pNA.AcOH,Cbo-D-Nleu-Gly-Arg-pNA.AcOH, Cbo-D-Nleu-Sar-Arg-pNA.AcOH,Cbo-D-Nval-Gly-Arg-pNA.AcOH, Cbo-D-CHA-Gly-Arg-pNA.AcOH,Cbo-D-CHA-Sar-Arg-pNA.AcOH, Cbo-D-CHT-Gly-Arg-pNA.AcOH,Cbo-D-CHT-Sar-Arg-pNA.AcOH, CH₃ SO₂ -D-Nleu-Gly-Arg-pNA.AcOH,isobutoxy-CO-D-Nleu-Gly-Arg-pNA.AcOH, BOC-D-Leu-Gly-Arg-pNA.HBr,4-MeO-Cbo-D-Leu-Gly-Arg-pNA.HBr, CH₃ O-CO-D-CHA-Gly-Arg-pNA.AcOH, C₂ H₅O-CO-D-CHA-Gly-Arg-pNA.AcOH, CH₃ SO₂ -D-CHA-Gly-Arg-pNA.AcOH,4-Me-Cbo-D-Leu-Gly-Arg-pNA.AcOH, 4-Cl-Cbo-D-Leu-Gly-Arg-pNA.AcOH,BOC-D-(α)-AOA-Gly-Arg-pNA.AcOH.